Steinbeck explores the theme of loneliness and isolation through specific character interactions. For instance, Candy, Crooks, and Lennie all exemplify the experience of alienation.

Candy is an aging and disabled ranch hand who has been relegated to cleaning the bunkhouse. Due to his age and physical disability, he cannot participate with the other men in ranch work and is excluded by the men when they visit the town. Steinbeck uses Candy’s old dog to parallel Candy’s own advanced age and diminished ability on the farm. Despite Candy’s reservations, the other ranch hands advocate for killing Candy’s dog, whom they believe is too old to be useful anymore. The ranch hands view Candy as being just as useless as his old dog, representing how the farm treats its own members once they've outlived their purpose. The men can exercise the power of putting the dog “out of its misery,” which is something they cannot exercise over Candy.

Crooks embodies the isolation and loneliness that arise from racial differences. As the only African American on the ranch, Crooks is ostracized and mistreated. At the time of the Great Depression, when Of Mice and Men is set, African Americans experienced a great deal of racism. Crooks exhibits an understanding of this; he realizes how and why he has been alienated from the other men. When Crooks and Curley’s wife get into a disagreement, Crooks’s belief that he can partake in the dream of owning land is dashed. He is quickly broken down again by Curley’s wife’s threats against him. Crooks sees that living in isolation is safer than interacting with the white men and women, whom he obviously cannot trust, because they will inevitably mistreat him.

Lennie faces the fear and stigma attached to having a mental disability, and we see the reverberations of this alienation throughout the novella. Although Lennie is cared for by George, he is misunderstood by many who encounter him, such as Curley’s wife. Lennie doesn’t wish to hurt anyone, but he lacks the social knowledge and physical control to avoid doing so. Lennie is left out of activities such as going to the town with George and the other men. However, Lennie also stands as a foil to loneliness through his absolute loyalty and friendship to George.