The Autobiography of Miss Jane Pittman begins with a note from the editor, who is a local schoolteacher near the plantation where Jane Pittman lives. He has long been trying to hear her story, and, beginning in the summer of 1962, she finally tells it to him. When her memory lapses, her acquaintances help fill in the spaces. The recorded tale, with editing, then becomes The Autobiography of Miss Jane.

Jane Pittman is born into slavery on a plantation somewhere in Louisiana. Jane is called "Ticey" during her days as a slave and has no parents; her mother died as a result of a beating when Jane was a child, and Jane did not know her father. Until she is around nine, Jane works in the Big House caring for the white children. One day toward the end of the war, some fleeing confederate soldiers arrive, followed soon after by some union soldiers. While being served water by Jane, one Union soldier named Corporal Brown tells Jane that she will soon be free and can then visit him in Ohio. He tells her to change her name and offers her that of his daughter, Jane Brown. After the soldiers leave, Jane refuses to answer when her mistress calls her "Ticey." The mistress later beats Jane until she bleeds, but Jane insists that her name is now Jane Brown. Because of her obstinacy, Jane is sent to work in the fields.

On the day of the Emancipation Proclamation, Jane's master frees them all. On the same day, Jane leaves the plantation with a group of ex-slaves. They have no idea where they are going, but a woman named Big Laura leads the way. Jane wants to go to Ohio to find Corporal Brown. The first morning away, a group of "Patrollers," local white trash who used to hunt slaves, comes upon them and kills everyone but Jane and a very young boy Ned, whom they did not find. Jane and Ned then continue on their own, still headed for Ohio. They meet many characters on their trip, all of whom tell Jane that Ohio is too far and that she should go back to her plantation. Jane's obstinacy persists for a few weeks until she and Ned are completely exhausted from walking. Finally they catch a ride with a poor white man named Job who lets them sleep at his house and takes them the next day to a plantation run by Mr. Bone. Mr. Bone offers Jane a job, but only pays her the reduced rate of six dollars a month (minus fifty cents for Ned's schooling) because she is so young. Jane and Ned get a cabin and after one month on the job, Mr. Bone raises her pay to ten dollars because she is doing as much work as the other women.

Life on Mr. Bone's plantation initially is good with a colored schoolteacher and a political scene still monitored by Republicans from the north. Then the original owner of the plantation, Colonel Dye, buys it back (with money borrowed from Yankees). Life reverts back to almost how it was before slavery, with segregation and violence against blacks who step out of line. The blacks start fleeing north because of the worsening conditions. Initially the whites do not care, but soon they try to stop the flight. Ned, who is now almost seventeen, joins a committee that helps blacks leave. Colonel Dye warns Jane that Ned must stop, but when he will not, Ku Klux Klan members arrive at Jane's house. Ned is not home when they come and is able to flee the plantation later that night. Jane does not want to leave her secure life, so they separate with sadness. Ned goes to Kansas, gets an education, and eventually joins the U.S. Army to fight in Cuba. Jane soon marries Joe Pittman (without an official ceremony). Despite Colonel Dye's attempts to keep them, Joe and Jane soon move to a ranch near the Texas-Louisiana border where Joe has found a job breaking horses.

Joe and Jane live at the new ranch for many years, but as they age Jane becomes increasingly worried about Joe getting hurt in his work. One of her recurring dreams depicts him being thrown from a horse. Soon after, Jane sees a black stallion in a corral that is the horse from her dream. She tries to get Joe not to ride it, even consulting a Creole voodoo woman, but after the horse escapes (because Jane frees it), Joe is killed trying to recapture it. After a few more years, Jane moves to another part of Louisiana with a fisherman, who suddenly leaves, and she is left all alone.

Ned soon moves back to where Jane is, and he brings his wife, Vivian, and three young children. He buys a house and starts building a school. At the school, he teaches ideas about the political rights of blacks as well as basic subjects. The local whites fear Ned's rhetoric, and therefore they hire a Cajun that Jane knows, Albert Cluveau, to shoot Ned, which Cluveau does. After Ned's death, Jane tells Cluveau that the chariot of hell will come for him and Cluveau later dies a fearful, painful death.

Jane then goes to live on the Samson plantation. Robert Samson runs the plantation with his wife, Miss Amma Dean. They have one son, Tee Bob, although Robert Samson had another son, Timmy, with a black woman on the plantation, Verda. Timmy looks and acts more like Robert than does Tee Bob, and the two boys are close friends even though Robert and Miss Amma Dean still expect Timmy to be subservient to his brother since Timmy is black. After the white overseer, Tom Joe, severely beats Timmy in response to Timmy's obstinacy, Robert Samson gives Timmy money and tells him to leave the plantation.

Later in life, Tee Bob falls in love with the Creole schoolteacher, Mary Agnes LeFarbre, who appears almost white. His friends and family remind him that a white man cannot love a black woman, but one night he goes to her house and asks her to marry him anyhow. After she tells him that he is not thinking straight, he returns home and commits suicide. Tee Bob's stepfather intervenes after the suicide so that Mary Agnes is not imprisoned or killed in revenge for Tee Bob's death. In a conversation with Jane, he describes that they all killed Tee Bob because of their adherence to racial regulations beyond which Tee Bob could see.

In the final chapter of the book, Jane describes a boy named Jimmy Aaron, whom the whole plantation hopes will become the "one" who will save them all. Eventually, Jimmy gets involved in the civil rights movement. After several years away from the plantation, he returns home and plans an act of civil disobedience followed by a protest at the courthouse. First a young girl is arrested for drinking from a white water fountain. On the day that they all are to march to the courthouse in protest, however, Jimmy is shot dead. The crowd who was planning to march had already gathered when they hear the news. With the assistance of one young black man, Jane bravely encourages the people to march and takes the lead even though Jimmy is already dead.