Book I

My Antonia is narrated by Jim Burden, a young white man who in the course of the novel goes to college, leaves his home of Black Hawk, Nebraska, and later becomes a lawyer. He tells the story of the titular character, Antonia Shimerda, the eldest daughter of a family of Bohemians living in a little farm community to the west of Black Hawk. Jim, who is orphaned at the beginning of the novel and sent to live with his grandparents, spends most of his childhood playing with Antonia, his neighbor, whom he teaches to read.

Jim and Antonia also spend time with Peter, one of the two Russians who live nearby and work a small farm with chickens on it. When Peter's friend Pavel grows ill, Jim and Antonia finally hear the story of how, when they lived in Russia, their sledge was run down by wolves, and everyone in their caravan was killed. Peter and Pavel fled Russia in shame. Unable to support themselves, they fall into debt in Nebraska and lose their farm. Jim and Antonia never see them again.

In January, Mr. Shimerda kills himself after a long battle with homesickness. His family, Antonia included, must fend for themselves. Upon moving into a new log cabin their neighbors helped to build (the Shimerdas were living in a roomy cave before), the mother buys a windmill on credit, preparing herself for the long planting and harvesting seasons. Meanwhile, Jim starts going to the country school and sees less of Antonia. Mostly, they spend time together in the summer.

Book II

Jim relates how he and his grandparents move from the farm to Black Hawk and transition from being farm people to town people . Their neighbors the Harlings have a small farm of their own, and Jim regularly spends time with the Harling children. Most of their country friends stop in to visit on their way to and from various destinations. Antonia visits often and becomes a servant to the Harlings. One day, their friend Lena Lingard, who grew up in a Norwegian settlement, comes to town. Soon, everyone settles into a routine.

Ostensibly, this book of the novel is about servants, and after Jim tells about Antonia becoming a servant, he introduces readers to a blind piano player named Samson who plays at Mrs. Gardener's hotel. One weekend, when Mrs. Gardener is out of town, Jim, Antonia, Lena, and Tiny all dance. Spring comes, and with it dances, roller-skating, and more. Lena gets involved with a young man named Sylvester Lovett, who leads her on and then marries a widow. Jim begins to feel contempt for certain town people like Lovett.

Antonia goes to work for Wick Cutter, the money-lender who destroyed Peter back in Book I. As soon as she starts her new job, she starts partying like Lena does. Jim often fantasizes about Lena and kissing her, but never fantasizes about Antonia like that, and they never enter into a romantic relationship. She's dating Larry Donovan, a kind of "professional ladies' man," and still thinks of Jim as a boy because he's four years younger than her. She's proud of his success in school, and he's proud to be seen walking around town with her.

In part because Antonia and Jim are so close and in part because she has no one else to turn to in this case, Antonia asks Jim to go sleep in her room at the Cutter place while she stays with Jim's grandmother. Mr. and Mrs. Cutter are out of town then, and Antonia doesn't feel safe alone in the house. One night, Mr. Cutter comes home alone and sits down on Antonia's bed, only to fly into a rage when he finds Jim sleeping there. This proves that Antonia has been abused by Mr. Cutter, but no charges are brought against him. Antonia moves out, with Jim's help.

Book III

Jim leaves Black Hawk to go to university in Lincoln, Nebraska. He doesn't come back that first summer, instead staying in Lincoln to study Greek and to build a friendship with his teacher, the sickly Gaston Cleric. Then, in his sophomore year, Lena Lingard comes to visit him. She lives in Lincoln now and owns a little dressmaking shop. She tells him Antonia is still dating Larry, that cad, and that she won't hear a bad word about Larry, though there are plenty of things Lena could say about him. She sighs, then asks Jim if he'd like to go to a show together sometime. As it turns out, Lincoln has a vibrant theatre scene, and Jim and Lena spend a lot of time together, watching shows and eating breakfast on Sundays. Then Cleric unexpectedly offers to take Jim East, and he leaves Lena behind in Lincoln after some consideration.

Book IV

Jim returns home for vacation the summer before entering law school. He knows, even before he arrives, that Larry Donovan abandoned Antonia, leaving her unwed and pregnant. He's extremely disappointed in her, especially considering how well Lena turned out. He then tells the story of a girl named Tiny, who went West, became the proprietor of a lodging house, moved to the Yukon in search of gold, laid claim to some land, spent years cooking for miners and amassing a small fortune, then moved to San Francisco and lived in luxury.

One day, while getting his grandparents' photo taken, Jim decides to see Antonia. He goes first to Mrs. Harling, who has always been good to Antonia, and then to the Widow Steavens, who cared for Antonia when the baby was born. The widow tells Jim about how Larry Donovan sent a letter to Antonia asking her to marry him, only to abandon her almost as soon as she arrived in the city. Antonia had no choice but to return to Black Hawk in shame and work for her keep. She had the baby one night after coming in from the fields and has by all accounts been a very good mother.

Finally, Jim visits Antonia at her family farm. Together, they walk out into the fields, where Jim finally confesses to Antonia that he would've liked her to be his sweetheart, or his wife, or even his mother or sister—"anything that a woman can be to a man." He promises to come back and visit her so she won't get lonely.

Book V

Twenty years later, Jim finally keeps his promise and visits Antonia. This visit is precipitated by a trip to San Francisco, in which Jim sees Lena and Tiny and learns that Antonia has about ten or eleven children and is now called Antonia Cuzak. He stops in Nebraska on his way back east and goes to Antonia's farm, where he meets her children and talks with her a while. Her husband isn't home then, but Jim gets a good picture of Antonia's life with him on the farm, learning the names and ages of their children and walking around their large, bountiful orchard. Jim joins Antonia and the children for dinner, then listens to one of the boys, Leo, play the organ. Jim is struck by Leo's liveliness, which stems from Antonia's own large, generous heart.

Jim winds up spending the night and is still there when Antonia's husband Cuzak returns the next afternoon. Cuzak brings some Bohemian papers, and he and Jim speak briefly of a famous singer named Maria Vasak, who has broken her leg. Antonia then relates the story of Wick Cutter: how he grew old, bought a pistol, killed his wife, and then shot himself to cheat Mrs. Cutter out of the money she would've inherited after his death. Jim has to admit that in all his years as a lawyer, he has never heard of anything so cruel. Cuzak himself is a much simpler man: the son of a cobbler, he met Antonia while visiting his cousin Anton Jelinek and immediately asked her to marry him. They've been together ever since.

Jim leaves the next night and spends a disappointing day in Black Hawk, where he hardly knows anyone anymore. He happens to stumble onto one of the old roads from his childhood and takes comfort in the knowledge that no matter how far apart he and Antonia are, they will always share "the precious, the incommunicable past."