Near the Salinas River in California, George Milton and Lennie Small, two migrant farm workers, walk toward a ranch. George is quick and smart, while Lennie is huge, strong, and mentally disabled. As they make their way to the ranch, George often whines that his life would be so much easier without Lennie. But when Lennie offers to leave him, George refuses. They bed down for the night, and George describes the farm that he and Lennie one day dream of owning together. George also reminds Lennie of the trouble Lennie got into at their last ranch and tells Lennie that if he gets into trouble again, he should hide at this spot where they're sleeping.
George and Lennie arrive at the ranch the next morning. There they meet Candy, an old handyman with only one hand, and the boss, who questions George and Lennie about their skills. The boss is skeptical when George answers for Lennie, but gives them work despite his suspicions. The men also meet Curley, the boss's ill-tempered and violent son, and, later, Curley's sexy wife, who likes to flirt with the ranch hands. Finally, George and Lennie meet Slim and Carlson. Slim's dog has just given birth, and Carlson wants to replace Candy's old, useless dog with one of the puppies.
After their first day of work, Slim gives a puppy to an overjoyed Lennie, and George tells Slim about what happened to him and Lennie at the ranch in Weed: Lennie touched a woman's dress and the woman accused him of rape. Slim voices his appreciation of the deep and selfless friendship between George and Lennie. Carlson, meanwhile, convinces Candy to let him shoot his dog. Carlson takes the dog outside and shoots it in the back of the head to make sure it doesn't suffer, but Candy still seems upset. When Lennie once again asks George to describe the farm they'll someday own, Candy overhears and asks if he can help them buy it. A few minutes later, Curley enters and the other men tease him about his wife. Curley mistakenly thinks Lennie is also laughing at him, so he punches Lennie. Lennie doesn't fight back until George says it's all right. Then Lennie catches Curley's hand and breaks it.
All of the men except Crooks, Lennie, and Candy go to visit a brothel on Saturday night. Though at first Crooks, a black man, refuses to talk to the white men, after a while they all end up talking about Lennie and George's farm. Crooks claims it will never work out. The conversation ends when Curley's wife enters. She mocks the men as weak and says she's so lonely that she's stooped to talking to them. When they tell her to leave, she threatens to have Crooks lynched.
The next afternoon, Lennie sits alone, consoling himself after accidentally killing his puppy. Curley's wife, lonely and desperate, sits beside him. She tells him of her failed dream of being an actress and offers to let Lennie touch her hair. But Lennie strokes it too hard. She tries to push him away. Lennie panics and shakes her, breaking her neck. Lennie flees to the spot by the river.
Candy discovers Curley's wife's body and alerts George. Curley leads a group of men to lynch Lennie, but George directs the men to the wrong place and finds Lennie first. As he once more describes the farm they're going to own and Lennie loses himself in imagination, George shoots Lennie in the back of the head with Carlson's gun. The men come running. George says he killed Lennie in a struggle for Carlson's gun. Slim tells George he did what he had to do.