The novel begins on a train trip in which the narrator and a childhood friend, Jim Burden, share words about the past. Their conversation is a reminiscence about their small hometown of Black Hawk, Nebraska, and a Bohemian girl they both remember named Ántonia Shimerda. Jim has written a memoir about her and the narrator expresses interest in reading the manuscript. Jim delivers it to her, changing the title to My Ántonia, and thus indicating that the script will be a personal story. With this introduction, Ántonia Shimerda's story begins and is marked by the changes in the seasons of both life and the prairie.
The reader travels back in time to when Jim Burden's parents die and his Virginia relatives send the 10-year-old Jim to his grandparents, who live on a Nebraska farm. During his train trip, Jim learns of an immigrant family that is also traveling to Black Hawk. When they reach their destination, Otto Fuchs, a cowboy, picks up Jim and Jake Marpole, a hired hand who has accompanied Jim; leaving the station, Jim sees the immigrant family, looking huddled and lost.
Jim meets his kindly grandparents and by the next day he is already appreciating a whole new world of earth, sun, and unending prairie. The following Sunday, Grandmother, Otto, and Jim take provisions to their new neighbors, the immigrant Shimerda family, and find that they are living in a lean-to that fronts a cave. Jim plays with Yulka and Ántonia and agrees to help Ántonia learn English. He also takes his first long pony ride in the autumn colors of Nebraska. But, unlike Jim, the Shimerdas stay close to their farm, believing that the townspeople cannot be trusted, and allowing Ántonia to faithfully see Jim for her English lessons.
At this point there is a side story about Pavel and Peter, two Russians whom Ántonia's father has befriended. On the way home Jim gives Ántonia a reading lesson on a bank near a badger hole. Ántonia explains that in Bohemia the badger is revered; they also rescue a frail green insect, reminding Ántonia of stories from "home." They meet Mr. Shimerda who has killed three rabbits, and he says he will make Ántonia a hat and someday give Jim his gun, a prized possession from Bohemia. His sad smile is a poignant foreshadowing of the future. Ántonia is feeling superior to Jim because she is four years older, but one day when he kills a huge rattlesnake Ántonia tells everyone of their adventure and brags about Jim's role. Now they seem to be more equal. This is only the beginning of their shared memories.
In the fall, the Russians get into trouble with Wick Cutter, Black Hawk's moneylender, and must pay a huge bonus on an overdue loan. When Pavel injures himself while building a barn, Mr. Shimerda, Ántonia, and Jim visit him and hear a grizzly story about why they left Russia. A few days later Pavel dies and Peter sells everything and goes away to cook at a construction camp. Their sad story reflects the harshness of the immigrant experience on the prairie.
Winter comes on and the first few weeks are bitter. Jim takes Ántonia and Yulka in a sled to the Russian's old house. On the way home, the elements are so severe that Jim gets sick and has to stay in with what today is called tonsillitis. These weeks are filled with family togetherness, popcorn, taffy, and storytelling. Jake tells Grandmother that the Shimerdas are hungry and living in wretched conditions, so they take Ántonia's family a big hamper of food. A few days later there is a tremendous snowstorm and the Burdens are forced to have "a country Christmas" and stay in with gingerbread cookies and homemade presents. They also send gifts to the Shimerdas. There are prayers and the reading of the Christmas Story from the gospels, culminating with an afternoon visit from Mr. Shimerda. He thanks the Burdens for their kindness and relaxes in the warm, family atmosphere. He crosses himself to pray and, though Jim is afraid his grandfather might be angry because he is conservative in religious matters, Grandfather says that the prayers of all good people are good.
Following Christmas the weather is better and Mrs. Shimerda and Ántonia visit the Burdens. Unlike her humble and grateful husband, Mrs. Shimerda complains that while her family struggles, the Burdens live in luxury, causing Grandmother Burden to give her a cooking pot. Meanwhile, Ántonia tells Jim that her father only came to America because her mother wanted Ambrosch to become rich.
In January, on Jim's eleventh birthday, a blizzard buries the prairie. It is during this time that the despondent Mr. Shimerda commits suicide, putting the barrel of a gun to his mouth and pulling the trigger. While arrangements are made about the body, Jim feels sad for the sensitive man and is hoping that Mr. Shimerda's soul is resting before its long journey back to Bohemia.
The next day Otto returns from Black Hawk with Anton Jelinek, a young Bohemian farmer, who has come to help his fellow immigrants. The coroner and priest arrive and a coffin is fashioned but Mr. Shimerda is denied burial in the Catholic or Norwegian cemetery. Mrs. Shimerda insists that he be buried at the corner of their land where future roads will eventually cross. They inter Mr. Shimerda and years later his grave is a little island with roads curving around it. Jim loves the quiet grave, and in the distant future it is here where he meets and talks with Ántonia.
In the spring, the Shimerdas are living in a new house their neighbors help build and Ántonia is working in the fields like a farm hand. Jim notices that she has become manly and coarse, Mrs. Shimerda remains suspicious and ungrateful, and Ambrosch is deceitful and sly. There is a rift between the two families over a horse collar but it is later healed by Grandfather.
When Jim has been with his grandparents three years, they decide to move into Black Hawk. Here Jim longs for the prairie and misses his friendship with Ántonia and the farmhands. His grandmother arranges for a neighbor, Mrs. Harling, to engage Ántonia as her hired girl. This marks the beginning of Ántonia's experiences in town. She works well for the Harlings, but she is also drawn to a dance pavilion with a group of other hired girls. Among them are Lena Lingard, a dressmaker, and Tiny Soderball, who works at the Boys' Home Hotel. Lena has a dubious reputation and is flirtatious and beautiful. As Jim continues to spend time with Ántonia, by summer her social life is interfering with her job and she is fired. So she goes to work for Wick Cutter, the notorious moneylender and womanizer.
By now a bored Jim is sneaking out at night to dances and seeing Lena who has let him kiss her. He begins to dream sensuous dreams of her but wishes his dream girl were Ántonia. When his grandmother discovers he is sneaking out, Jim is contrite and promises not to do it anymore. A summer picnic is an opportunity for one last idyllic day spent with Ántonia and Lena where Ántonia scolds him for flirting with Lena, telling him he has a greater future than life in Black Hawk. The day ends with a symbolic image of a plow, bold and black against the setting sun.
Meanwhile, Ántonia is suspicious of a trip planned by Wick Cutter and her misgivings prove to be correct. She convinces Jim to spend the night at the Cutters' house and Wick returns in an attempt to seduce Ántonia. She leaves the Cutter employ and once again is engaged by the Harlings.
Jim spends the summer studying for exams to get into the state university. In the fall, he leaves for Lincoln and the university, but no matter how much he studies he finds his life on the prairie and his friendships interfering with his concentration. Lena moves to Lincoln, and she and Jim begin a social life that includes attending plays together, most notably Dumas' Camille. This leads Jim to neglect his studies and his Latin teacher, Gaston Cleric, suggests that Jim transfer to Harvard University where Cleric is taking a position. Jim is now nineteen and his grandfather permits him to go to the East. Before he leaves, Lena tells him that Ántonia is engaged to a railroad man, Larry Donavan.
The friends go their separate ways. Jim leaves for Harvard and Ántonia goes to Denver to marry Larry Donovan. Lena opens a dressmaking shop in San Francisco and Tiny Soderball eventually ends up in Alaska where she becomes wealthy. Jim assumes that Ántonia is fine, but he later finds out Donovan deceived her, leaving her pregnant and unmarried. Ántonia returns to Black Hawk, has her baby, and works in the fields once again. On a return visit Jim spends a day with Ántonia and she shows him her baby. She seems somber but settled and Jim tells her how much she has become a part of him. He holds also the memories of her father who lives on in both their hearts. Jim believes this will be the last time they will see each other and he tries to memorize the prairie, the fields, and the tall grass, wishing he were a boy again with a laughing Ántonia running beside him.
Twenty years later, Jim Burden sees Ántonia on a trip to Black Hawk. She has married well to Anton Cuzak, a cousin of Anton Jelinek. They have many children and Jim spends an evening looking at old photographs with them and hearing the stories of the past from Ántonia's children. Meeting her husband, Jim finds that Cuzak already seems to know him because Ántonia has described him so well to her whole family. Happy and contented, Ántonia is rooted in the prairie and the Nebraska farmland. When Jim leaves, he promises to return and take the Cuzak boys hunting the next summer.
Reflecting on the past, Jim realizes that many of his old friends have died or moved away, but he still has memories and longings for the prairie. He remembers the train trip so long ago where he first saw the frightened and small immigrant girl, Ántonia. Now she seems larger than life, like the plow against the sunset, acclimated to the prairie land. In contrast, Jim seems rootless, traveling and adrift in life. He vows to return to the prairie where he felt a peace and contentment that eludes him in the East, and where Ántonia and her family share his past.