Arthur Shelby, a Kentucky farmer and slaveowner, is forced by debt to sell two slaves — Uncle Tom and Harry, the young son of his wife's servant Eliza — to a trader named Haley. Eliza hears the discussion, warns Tom and his wife, and runs away with her child, followed by Haley, who is prevented from catching her when she crosses the Ohio River and is aided by helpful citizens. Haley meets two slave-catchers who agree to pursue Eliza and Harry. Meanwhile Tom refuses to run away and is taken by Haley toward New Orleans. Before leaving Kentucky, however, Haley buys several more slaves, and one of them (a young mother whose infant Haley sells without her knowledge) commits suicide.
Some time later, Eliza's husband, George Harris, himself an escaped slave in disguise, discovers that Eliza is headed for Canada and sets out to find and join her. Meanwhile, Eliza and her son have been taken in by a Quaker family and are joined by George to prepare for the next stage of their escape.
Tom, on a Mississippi river boat, meets a little white girl named Eva St. Clare, is touched by her beauty and gravity, and rescues her from drowning. Eva's father buys Tom from Haley at Eva's request, and Tom accompanies the family (father, daughter, and cousin Ophelia) to their New Orleans home. There he meets Eva's mother, a spoiled and bigoted woman, and other slaves belonging to the household. He and Eva form a close relationship; by reading to Tom from his Bible, Eva herself grows to understand and love Christianity.
George and Eliza Harris, with their child and two other escaped slaves, are being driven to the next stop on their journey when pursuers overtake them. George wounds one with his pistol; the rest of the posse flees. Eliza persuades the others to bring the wounded man with them to be treated.
Back in New Orleans, Tom has been given the responsibility of marketing for the St. Clare household. St. Clare writes a letter to Tom's wife in Kentucky, informing her of Tom's whereabouts and well-being. St. Clare also buys a young slave girl called Topsy and "gives" her to Ophelia to raise.
Back in Kentucky, Tom's wife, Chloe, convinces Mrs. Shelby that Chloe should be hired out to a confectionary baker in Louisville and her wages saved to buy Tom's freedom. The Shelbys' son, George, writes back to Tom with this news.
After two years, it becomes apparent to Tom — and soon to others — that Eva is terminally ill. Her father refuses to see the truth of this. But after a visit from St. Clare's plantation-owner brother and the brother's young son, Eva's condition worsens, and St. Clare finally must accept the knowledge that she is dying. As death approaches, Eva touches the hearts of all around her, even Topsy, with her sweet Christian acceptance, and when she dies, everyone mourns her. Tom's influence at this point brings St. Clare almost to belief in Christ, and the man promises Tom his freedom, signs Topsy over to Ophelia legally, and begins to make provisions to protect all of his slaves from sale, should something happen to him. But then St. Clare is killed suddenly, and his wife sells most of his servants, including Tom.
Tom's new owner is Simon Legree, a plantation owner, who also buys two women, one intended as the sexual slave of Legree's black overseer Sambo, the other (a 15-year-old named Emmeline) for Legree himself. They are taken to the man's run-down plantation among the swamps. Tom is set to picking cotton, and he tries to make the best of his position by prayer and hope. He meets Cassy, Legree's black concubine, and learns her horrifying story. Tom is whipped mercilessly for attempting to help his fellow slaves, and Legree vows to break his spirit or kill him. Cassy does her best to use her influence to save Tom.
Back in the Midwest, Tom Loker has warned the Quakers that Eliza and her family are being sought at the Lake Erie port where they expect to cross into Canada, so Eliza disguises herself and is not recognized. She, George, and Harry cross into freedom.
But Tom, in the months that follow his beating, loses heart and nearly his faith, until at the lowest ebb of his life he is given the grace to prevail in spirit against Legree's torture. He brings his own spiritual strength to the other slaves, and Cassy devises a way for her and Emmeline to escape. The two women hide in Legree's own garret while the man searches the swamps for them. Legree questions Tom, who knows their plan but refuses to tell. Legree has Sambo and the other overseer whip Tom until he is near death; finally Legree gives up, and the dying Tom forgives him and the two men who whipped him.
George Shelby, arrived to buy Tom's freedom, is in time only to hear his last words. But Cassy and Emmeline have made good their escape, and they meet George on the riverboat going north. Another lady on the boat reveals that she is George Harris's sister, and Cassy recognizes that George's wife Eliza is her own daughter. The two, with Emmeline, go to Canada and find George, Eliza, and their children; they all eventually go to France, return, and plan to emigrate to Liberia. Meanwhile George Shelby returns to his farm, where his father has died, breaks the news to Chloe of Tom's death, and frees all his slaves, telling them to remember that they owe their freedom to the influence of Uncle Tom.