The novel is divided into eight parts. Its epigraph is "Vengeance is mine; I will repay", from Romans 12:19, which in turn quotes from Deuteronomy 32:35.
The novel begins with one of its most often-quoted lines: "Happy families are all alike; every unhappy family is unhappy in its own way."
The novel opens with a scene that introduces Prince Stepan Arkadyevich Oblonsky ("Stiva"), a Moscow aristocrat and civil servant who has been unfaithful to his wife, Princess Darya Alexandrovna ("Dolly"). Dolly has discovered his affair with the family's governess, and the household and family are in turmoil. Stiva informs the household that his married sister, Anna Arkadyevna Karenina, is coming to visit from Saint Petersburg in a bid to calm the situation.
Meanwhile, Stiva's childhood friend, Konstantin Dmitrievich Levin ("Kostya"), arrives in Moscow with the aim of proposing to Dolly's youngest sister, Princess Katerina Alexandrovna Shcherbatskaya ("Kitty"). Levin is a passionate, restless, but shy aristocratic landowner who, unlike his Moscow friends, chooses to live in the country on his large estate. He discovers that Kitty is also being pursued by Count Alexei Kirillovich Vronsky, an army cavalry officer.
Whilst at the railway station to meet Anna, Stiva bumps into Vronsky who is there to meet his mother, the Countess Vronskaya. Anna and Vronskaya have traveled and talked together in the same carriage. As the family members are reunited, and Vronsky sees Anna for the first time, a railway worker accidentally falls in front of a train and is killed. Anna interprets this as an "evil omen". Vronsky, however, is infatuated with Anna, and donates two hundred roubles to the dead man's family, which impresses her. Anna is also uneasy about leaving her young son, Sergei ("Seryozha"), alone for the first time.
At the Oblonsky home, Anna talks openly and emotionally to Dolly about Stiva's affair and convinces her that Stiva still loves her despite the infidelity. Dolly is moved by Anna's speeches and decides to forgive Stiva.
Kitty, who comes to visit Dolly and Anna, is just eighteen. In her first season as a debutante, she is expected to make an excellent match with a man of her social standing. Vronsky has been paying her considerable attention, and she expects to dance with him at a ball that evening. Kitty is very struck by Anna's beauty and personality and becomes infatuated with her just as Vronsky. When Levin proposes to Kitty at her home, she clumsily turns him down, believing she is in love with Vronsky and that he will propose to her, and encouraged to do so by her mother, who believes Vronsky would be a better match (in contrast to Kitty's father, who favors Levin).
At the big ball Kitty expects to hear something definitive from Vronsky, but he dances with Anna instead, choosing her as a partner over a shocked and heartbroken Kitty. Kitty realizes that Vronsky has fallen in love with Anna and has no intention of marrying her, despite his overt flirtations. Vronsky has regarded his interactions with Kitty merely as a source of amusement and assumes that Kitty has acted for the same reasons. Anna, shaken by her emotional and physical response to Vronsky, returns at once to St. Petersburg. Vronsky travels on the same train. During the overnight journey, the two meet and Vronsky confesses his love. Anna refuses him, although she is deeply affected by his attentions to her.
Levin, crushed by Kitty's refusal, returns to his estate, abandoning any hope of marriage. Anna returns to her husband, Count Alexei Alexandrovich Karenin, a senior government official, and her son, Seryozha, in St. Petersburg. On seeing her husband for the first time since her encounter with Vronsky, Anna realizes that she finds him unattractive, though she tells herself he is a good man.
The Shcherbatskys consult doctors over Kitty's health, which has been failing since Vronsky's rejection. A specialist advises that Kitty should go abroad to a health spa to recover. Dolly speaks to Kitty and understands she is suffering because of Vronsky and Levin, whom she cares for and had hurt in vain. Kitty, humiliated by Vronsky and tormented by her rejection of Levin, upsets her sister by referring to Stiva's infidelity, saying she could never love a man who betrayed her. Meanwhile, Stiva visits Levin on his country estate while selling a nearby plot of land.
In St. Petersburg, Anna begins to spend more time in the inner circle of Princess Elizaveta ("Betsy"), a fashionable socialite and Vronsky's cousin. Vronsky continues to pursue Anna. Although she initially tries to reject him, she eventually succumbs to his attentions and begins an affair. Meanwhile, Karenin reminds his wife of the impropriety of paying too much attention to Vronsky in public, which is becoming the subject of gossip. He is concerned about the couple's public image, although he believes mistakenly that Anna is above suspicion.
Vronsky, a keen horseman, takes part in a steeplechase event, during which he rides his mare Frou-Frou too hard—his irresponsibility causing him to fall and break the horse's back. Anna is unable to hide her distress during the accident. Before this, Anna had told Vronsky that she is pregnant with his child. Karenin is also present at the races and remarks to Anna that her behaviour is improper. Anna, in a state of extreme distress and emotion, confesses her affair to her husband. Karenin asks her to break it off to avoid further gossip, believing that their marriage will be preserved.
Kitty and her mother travel to a German spa to recover from her ill health. There, they meet the wheelchair-bound Pietist Madame Stahl and the saintly Varenka, her adopted daughter. Influenced by Varenka, Kitty becomes extremely pious, but becomes disillusioned by her father's criticism when she learns Madame Stahl is faking her illness. She then returns to Moscow.
Levin continues working on his estate, a setting closely tied to his spiritual thoughts and struggles. He wrestles with the idea of falseness, wondering how he should go about ridding himself of it, and criticising what he feels is falseness in others. He develops ideas relating to agriculture, and the unique relationship between the agricultural labourer and his native land and culture. He comes to believe that the agricultural reforms of Europe will not work in Russia because of the unique culture and personality of the Russian peasant.
When Levin visits Dolly, she attempts to understand what happened between him and Kitty and to explain Kitty's behaviour. Levin is very agitated by Dolly's talk about Kitty, and he begins to feel distant from Dolly as he perceives her loving behaviour towards her children as false. Levin resolves to forget Kitty and contemplates the possibility of marriage to a peasant woman. However, a chance sighting of Kitty in her carriage makes Levin realize he still loves her. Meanwhile, in St. Petersburg, Karenin refuses to separate from Anna, insisting that their relationship will continue. He threatens to take away Seryozha if she persists in her affair with Vronsky.
When Anna and Vronsky continue seeing each other, Karenin consults with a lawyer about obtaining a divorce. During the time period, a divorce in Russia could only be requested by the innocent party in an affair and required either that the guilty party confessed—which would ruin Anna's position in society and bar her from remarrying in the Orthodox Church—or that the guilty party be discovered in the act of adultery. Karenin forces Anna to hand over some of Vronsky's love letters, which the lawyer deems insufficient as proof of the affair. Stiva and Dolly argue against Karenin's drive for a divorce.
Karenin changes his plans after hearing that Anna is dying after the difficult birth of her daughter, Annie. At her bedside, Karenin forgives Vronsky. However, Vronsky, embarrassed by Karenin's magnanimity, unsuccessfully attempts suicide by shooting himself. As Anna recovers, she finds that she cannot bear living with Karenin despite his forgiveness and his attachment to Annie. When she hears that Vronsky is about to leave for a military posting in Tashkent, she becomes desperate. Anna and Vronsky reunite and elope to Europe, leaving Seryozha and Karenin's offer of divorce.
Meanwhile, Stiva acts as a matchmaker with Levin: he arranges a meeting between him and Kitty, which results in their reconciliation and betrothal.
Levin and Kitty marry and start their new life on his country estate. Although the couple are happy, they undergo a bitter and stressful first three months of marriage. Levin feels dissatisfied at the amount of time Kitty wants to spend with him and dwells on his inability to be as productive as he was as a bachelor. When the marriage starts to improve, Levin learns that his brother, Nikolai, is dying of consumption. Kitty offers to accompany Levin on his journey to see Nikolai and proves herself a great help in nursing Nikolai. Seeing his wife take charge of the situation in an infinitely more capable manner than he could have done himself without her, Levin's love for Kitty grows. Kitty eventually learns that she is pregnant.
In Europe, Vronsky and Anna struggle to find friends who will accept them. Whilst Anna is happy to be finally alone with Vronsky, he feels suffocated. They cannot socialize with Russians of their own class and find it difficult to amuse themselves. Vronsky, who believed that being with Anna was the key to his happiness, finds himself increasingly bored and unsatisfied. He takes up painting and makes an attempt to patronize an émigré Russian artist of genius. However, Vronsky cannot see that his own art lacks talent and passion, and that his conversation about art is extremely pretentious. Increasingly restless, Anna and Vronsky decide to return to Russia.
In St. Petersburg, Anna and Vronsky stay in one of the best hotels, but take separate suites. It becomes clear that whilst Vronsky is still able to move freely in Russian society, Anna is barred from it. Even her old friend, Princess Betsy, who has had affairs herself, evades her company. Anna starts to become anxious that Vronsky no longer loves her. Meanwhile, Karenin is comforted by Countess Lidia Ivanovna, an enthusiast of religious and mystic ideas fashionable with the upper classes. She advises him to keep Seryozha away from Anna and to tell him his mother is dead. However, Seryozha refuses to believe that this is true. Anna visits Seryozha uninvited on his ninth birthday but is discovered by Karenin.
Anna, desperate to regain at least some of her former position in society, attends a show at the theatre at which all of St. Petersburg's high society are present. Vronsky begs her not to go, but he is unable to bring himself to explain to her why she cannot attend. At the theatre, Anna is openly snubbed by her former friends, one of whom makes a deliberate scene and leaves the theatre. Anna is devastated. Unable to find a place for themselves in St. Petersburg, Anna and Vronsky leave for Vronsky's own country estate.
Dolly, her mother the Princess Scherbatskaya, and Dolly's children spend the summer with Levin and Kitty. The Levins' life is simple and unaffected, although Levin is uneasy at the "invasion" of so many Scherbatskys. He becomes extremely jealous when one of the visitors, Veslovsky, flirts openly with the pregnant Kitty. Levin tries to overcome his jealousy, and briefly succeeds during a hunt with Veslovsky and Oblonsky, but eventually succumbs to his feelings and orders Veslovsky to leave in an embarrassing scene. Veslovsky immediately goes to stay with Anna and Vronsky at their nearby estate.
When Dolly visits Anna, she is struck by the difference between the Levins' aristocratic-yet-simple home life and Vronsky's overtly luxurious and lavish country estate. She is also unable to keep pace with Anna's fashionable dresses or Vronsky's extravagant spending on a hospital he is building. In addition, all is not quite well with Anna and Vronsky. Dolly notices Anna's anxious behaviour and her uncomfortable flirtations with Veslovsky. Vronsky makes an emotional request to Dolly, asking her to convince Anna to divorce Karenin so that the two might marry and live normally.
Anna has become intensely jealous of Vronsky and cannot bear when he leaves her, even for short excursions. When Vronsky leaves for several days of provincial elections, Anna becomes convinced that she must marry him to prevent him from leaving her. After Anna writes to Karenin, she and Vronsky leave the countryside for Moscow.
While visiting Moscow for Kitty's confinement, Levin quickly gets used to the city's fast-paced, expensive and frivolous society life. He accompanies Stiva to a gentleman's club, where the two meet Vronsky. Levin and Stiva pay a visit to Anna, who is occupying her empty days by being a patroness to an orphaned English girl. Levin is initially uneasy about the visit, but Anna easily puts him under her spell. When he admits to Kitty that he has visited Anna, she accuses him of falling in love with her. The couple are later reconciled, realising that Moscow society life has had a negative, corrupting effect on Levin.
Anna cannot understand why she can attract a man like Levin, who has a young and beautiful new wife, but can no longer attract Vronsky. Her relationship with Vronsky is under increasing strain, because he can move freely in Russian society while she remains excluded. Her increasing bitterness, boredom, and jealousy cause the couple to argue. Anna uses morphine to help her sleep, a habit she began while living with Vronsky at his country estate. She has become dependent on it. Meanwhile, after a long and difficult labour, Kitty gives birth to a son, Dmitri, nicknamed "Mitya". Levin is both horrified and profoundly moved by the sight of the tiny, helpless baby.
Stiva visits Karenin to seek his commendation for a new post. During the visit, Stiva asks Karenin to grant Anna a divorce (which would require him to confess to a non-existent affair), but Karenin's decisions are now governed by a French "clairvoyant" recommended by Lidia Ivanovna. The clairvoyant apparently had a vision in his sleep during Stiva's visit and gives Karenin a cryptic message that he interprets in a way such that he must decline the request for divorce.
Anna becomes increasingly jealous and irrational towards Vronsky, whom she suspects of having love affairs with other women. She is also convinced that he will give in to his mother's plans to marry him off to a rich society woman. They have a bitter row and Anna believes the relationship is over. She starts to think of suicide as an escape from her torments. In her mental and emotional confusion, she sends a telegram to Vronsky asking him to come home to her, and then pays a visit to Dolly and Kitty. Anna's confusion and anger overcome her and, in a parallel to the railway worker's accidental death in Part 1, she commits suicide by throwing herself under the carriage of a passing train.
Sergei Ivanovich's (Levin's brother) latest book is ignored by readers and critics and he joins the new pan-Slavic movement. Stiva gets the post he desired so much, and Karenin takes custody of Vronsky and Anna's baby, Annie. A group of Russian volunteers, including the suicidal Vronsky, depart from Russia to fight in the Orthodox Serbian revolt that has broken out against the Turks.
A lightning storm occurs at Levin's estate while his wife and newborn son are outdoors and, in his fear for their safety, Levin realizes that he does indeed love his son as much as he loves Kitty. Kitty's family is concerned that a man as altruistic as her husband does not consider himself to be a Christian.
After speaking at length to a peasant, Levin has a true change of heart, concluding that he does believe in the Christian principles taught to him in childhood and no longer questions his faith. He realizes that one must decide for oneself what is acceptable concerning one's own faith and beliefs. He chooses not to tell Kitty of the change that he has undergone.
Levin is initially displeased that his return to his faith does not bring with it a complete transformation to righteousness. However, at the end of the story, Levin arrives at the conclusion that despite his newly accepted beliefs, he is human and will go on making mistakes. His life can now be meaningfully and truthfully oriented toward righteousness.