Most of the book weaves in and out of two timelines. In the frame tale of the narrative present, Spiegelman interviews his father Vladek in the Rego Park neighborhood of New York City in 1978–79. The story that Vladek tells unfolds in the narrative past, which begins in the mid-1930s and continues until the end of the Holocaust in 1945.
In Rego Park in 1958, a young Art Spiegelman complains to his father that his friends have left him behind. His father responds in broken English, "Friends? Your friends? If you lock them together in a room with no food for a week, then you could see what it is, friends!"
As an adult, Art visits his father, from whom he has become estranged. Vladek has remarried to a woman called Mala since the suicide in 1968 of Art's mother Anja. Art asks Vladek to recount his Holocaust experiences. Vladek tells of his time in the Polish city of Częstochowa and how he came to marry into Anja's wealthy family in 1937 and move to Sosnowiec to become a manufacturer. Vladek begs Art not to include this in the book and Art reluctantly agrees. Anja suffers a breakdown due to postpartum depression after giving birth to their first son Richieu,[b] and the couple go to a sanitarium in Nazi-occupied Czechoslovakia for her to recover. After they return, political and anti-Semitic tensions build until Vladek is drafted just before the Nazi invasion. Vladek is captured at the front and forced to work as a prisoner of war. After his release, he finds Germany has annexed Sosnowiec and he is dropped off on the other side of the border in the German protectorate. He sneaks across the border and reunites with his family.
During one of Art's visits, he finds that a friend of Mala's has sent the couple one of the underground comix magazines Art contributed to. Mala had tried to hide it, but Vladek finds and reads it. In "Prisoner on the Hell Planet", Art is traumatized by his mother's suicide three months after his release from the mental hospital, and in the end depicts himself behind bars saying, "You murdered me, Mommy, and left me here to take the rap!" Though it brings back painful memories, Vladek admits that dealing with the issue in such a way was for the best.
In 1943, the Nazis move the Jews of the Sosnowiec Ghetto to Srodula and march them back to Sosnowiec to work. The family splits up—Vladek and Anja send Richieu to Zawiercie to stay with an aunt for safety. As more Jews are sent from the ghettos to Auschwitz, the aunt poisons herself, her children and Richieu to escape the Gestapo. In Srodula, many Jews build bunkers to hide from the Germans. Vladek's bunker is discovered and he is placed into a "ghetto inside the ghetto" surrounded by barbed wire. The remnants of Vladek and Anja's family are taken away. Srodula is cleared of its Jews, except for a group Vladek hides with in another bunker. When the Germans depart, the group splits up and leaves the ghetto.
In Sosnowiec, Vladek and Anja move from one hiding place to the next, making occasional contact with other Jews in hiding. Vladek disguises himself as an ethnic Pole and hunts for provisions. The couple arrange with smugglers to escape to Hungary, but it is a trick—the Gestapo arrest them on the train (as Hungary is invaded) and take them to Auschwitz, where they are separated until after the war.
Art asks after Anja's diaries, which Vladek tells him were her account of her Holocaust experiences and the only record of what happened to her after her separation from Vladek at Auschwitz and which Vladek says she had wanted Art to read. Vladek comes to admit that he burned them after she killed herself. Art is enraged and calls Vladek a "murderer".
The story jumps to 1986, after the first six chapters of Maus have appeared in a collected edition. Art is overcome with the unexpected attention the book receives and finds himself "totally blocked". Art talks about the book with his psychiatrist Paul Pavel, a Czech Holocaust survivor. Pavel suggests that, as those who perished in the camps can never tell their stories, "maybe it's better not to have any more stories". Art replies with a quote from Samuel Beckett: "Every word is like an unnecessary stain on silence and nothingness", but then realizes, "on the other hand, he said it".
Vladek tells of his hardship in the camps, of starvation and abuse, of his resourcefulness, of avoiding the selektionen—the process by which prisoners were selected for further labor or execution. Despite the danger, Anja and Vladek exchange occasional messages. As the war progresses and the German front is pushed back, the prisoners are marched from Auschwitz in occupied Poland to Gross-Rosen within the Reich and then to Dachau, where the hardships only increase and Vladek catches typhus.
The war ends, the camp survivors are freed and Vladek and Anja reunite. The book closes with Vladek turning over in his bed as he finishes his story and telling Art, "I'm tired from talking, Richieu, and it's enough stories for now." The final image is of Vladek and Anja's tombstone—Vladek died in 1982, before the book was completed.