George Milton - The protagonist, a small, quick-witted man who looks after his friend Lennie. Despite being intelligent, he is stuck working as an itinerant ranch hand. He dreams of one day owning a farm with Lennie.

Lennie Small - A large man with the mind of a child, possessing tremendous physical strength but little understanding of his own power. He loves petting soft things but often accidentally kills them. Lennie's intellectual disability makes him dependent on George.

Curley - The antagonist, the son of the ranch owner. A confrontational and aggressive young man with a Napoleon complex, he wears high-heeled boots and picks fights with larger men like Lennie to prove his masculinity. He is extremely possessive and jealous of his flirtatious wife.

Curley's Wife - The only female character, she remains nameless throughout the novella, reflecting how she is viewed as Curley's possession. Dissatisfied with her life, she flirts with the ranch hands, leading to tragic consequences.

Candy - An elderly ranch handyman, he lost his hand in a machine accident and worries about being fired due to his disability. He offers to contribute his life's savings to George and Lennie's dream of owning a farm.

Crooks - The lonely, disabled black stable buck who faces discrimination and has insight into the futility of George and Lennie's dream of a farm.

Slim - A skilled ranch hand who is respected by the others. He represents an unbiased perspective and serves as a truth-teller in the story.