Anansi Boys is the story of Charles "Fat Charlie" Nancy, a timid Londoner devoid of ambition, whose unenthusiastic wedding preparations are disrupted when he learns of his father's death in Florida. The flamboyant Mr. Nancy, in whose shadow Fat Charlie has always lived, died in a typically embarrassing manner by suffering a fatal heart attack while singing to a young woman on stage in a karaoke bar.
Fat Charlie is forced to take time off from the talent agency where he works and travel to Florida for the funeral. Afterwards, while discussing the disposal of Mr. Nancy's estate, Mrs. Callyanne Higgler, a very old family friend, reveals to Fat Charlie that the late Mr. Nancy was actually an incarnation of the West African spider god, Anansi, hence his name. The reason Charlie had apparently not inherited any divine powers was because they had been passed down to his hitherto unknown brother, whom she mentions can be contacted by simply sending an invitation via a spider. Charlie is skeptical, and on his return to England, largely forgets what Mrs. Higgler had told him, until one night when he drunkenly whispers to a spider that it would be nice if his brother stopped by for a visit.
The next morning, the suave and well-dressed brother, going under the name of "Spider", visits Charlie, and is shocked to learn that their father had died. Immediately Spider steps through a picture to their childhood home. Charlie goes off to work, rather puzzled by Spider and his sudden disappearance.
Spider returns that night, stricken with grief that Anansi had died and that he (Spider) had been thoughtless enough not to notice. The two, to drown their sorrows, become uproariously drunk (at Spider's recommendation) on the proverbial trio of wine, women, and song. Although Charlie is not involved in most of the womanising or singing, he is drunk enough to sleep through much of the next day. Spider covers for Charlie's absence from his office at the Grahame Coats Agency by magically disguising himself as Charlie. In the process, Spider discovers Grahame Coats's long-standing practice of embezzling from his clients. Spider also steals the affection and virginity of Charlie's fiancée, Rosie Noah.
Spider, in the guise of Charlie, reveals his knowledge of the financial improprieties to Grahame Coats during a meeting which Grahame calls to fire Charlie. As a result, Grahame delays firing Charlie. When Grahame meets him next, he gives the real Charlie a large cheque and a holiday from work. With Charlie out of the office, Grahame Coats proceeds to alter the financial records to frame Charlie for the embezzlement. Embittered by the loss of his fiancée, Charlie uses his holiday to return to Florida, and requests help from Mrs. Higgler and three of her equally old and eccentric friends to expel Spider. Being themselves powerless in this matter, they instead send him to "the beginning of the world", an abode of ancient gods similar to his father, each of whom represents a species of animal. There, he encounters the fearsome Tiger, the outrageous Hyena, and the ridiculous Monkey, among others. None are willing to help the son of the trickster who had embarrassed them. Finally, he meets Bird Woman, who agrees to trade Charlie her help, symbolised by one of her feathers, in exchange for "Anansi's bloodline".
Meanwhile, in London, a swindled client, Maeve Livingstone, confronts Grahame Coats directly, having learned of the theft of her late husband's royalties. Grahame Coats murders her with a hammer and conceals her body in a hidden closet.
When Charlie returns to England, events begin to escalate. Charlie quarrels and scuffles with Spider; Charlie is taken in for questioning by the police for financial fraud at the Grahame Coats Agency; Spider reveals the truth to Rosie, who is angered by his treatment of her; birds repeatedly attack Spider; Grahame Coats escapes England for his estate and bank accounts in the fictional Caribbean country of Saint Andrews; and Maeve Livingstone's ghost begins haunting the Grahame Coats Agency building.
Maeve is contacted by her late husband, who advises her to move on to the afterlife. She refuses in favour of taking vengeance on Grahame Coats. Later, she meets the ghost of Anansi himself, who recounts a story to her. Once, Anansi reveals, the animal god Tiger owned all stories, and as a result, all stories were dark and violent; but Anansi tricked Tiger into surrendering the ownership of stories to Anansi, so stories now involve cleverness and skill rather than strength alone.
After he is attacked by flamingoes, Spider realises that something Charlie did is causing these attacks, and that he is in mortal peril. He spirits Charlie out of prison. They discuss matters in the course of fleeing from birds around the world, realising that giving away Anansi's bloodline implicates Charlie as well as Spider. Charlie is then returned to prison. He is eventually freed, and mentions the hidden room in Coats's office, where the police will find Maeve Livingstone's body.
Spider is swept away in a storm of birds, after which Bird Woman removes his tongue to prevent his use of magic, and delivers him to Tiger, Anansi's longtime enemy, who imprisons him. In spite of his helplessness, Spider manages to form a little spider out of clay, instructing it to go find help in the spider kingdom that Anansi and his descendants command. Though not as effective a hunter as Tiger, Spider can still fend him off for a little while, whereas Tiger is pleased to draw out the hunt, as it allows him to savour his long hoped-for revenge on Anansi and his brood.
Rosie and her mother have taken a consolation cruise to the Caribbean, where they unexpectedly meet Grahame Coats. They have not heard of the events in England, and so unsuspectingly walk into his trap and are locked in his basement.
Charlie goes searching for Callyanne Higgler to help him solve his problems. He looks for her in Florida, but Anansi's old friends tell him that Mrs Higgler has returned to the Caribbean country of Saint Andrews. They also reveal to him that it was another old lady, Mrs. Dunwiddy, who, annoyed with the young Fat Charlie, made a spell to separate his good side from his bad side, which then became Spider, meaning that Fat Charlie and Spider had once been one person. He finally finds her after a long search in Saint Andrews and is sent again to the beginning of the world. Charlie forces the Bird Woman to give back Anansi's bloodline in return for her feather. Meanwhile, Spider has managed to survive, Tiger having grown overconfident. When Tiger attempts a killing strike, the reinforcements summoned by Spider overwhelm him. At that point, Charlie rescues Spider and gives him back his tongue.
Tiger now takes possession of Grahame Coats's body and uses his blood-lust to manipulate him, intending to get revenge on Spider by killing Rosie and her mother. But the possession by Tiger makes Grahame Coats vulnerable to attacks from other spirits; Maeve Livingstone, having found Grahame Coats with the aid of Anansi's ghost, eliminates Coats in the real world and, satisfied, moves on to her afterlife.
Meanwhile, still at the beginning of the world, Charlie, having discovered his power to alter reality by singing a story, recounts the long tale of all that has gone before, humiliating Tiger to the point of retreat. Spider then collapses the cave entrance, sealing Tiger and Grahame Coats into the cave. Charlie weaves this event into his song, reinforcing it with his powers, such that Tiger is now well and truly trapped. Coats, turned into a Stoat, remains with Tiger as unwelcome (and eternally edible) guest.
In the end, Spider marries Rosie and becomes the owner of a restaurant. He is put constantly under pressure by Rosie's mother to have children, but (possibly to annoy her) never does. Charlie begins a successful career as a singer, marries police officer Daisy Day and has a son (Marcus). Old Anansi, resting comfortably in his grave, watches his two sons approvingly.