BACKGROUND: Low physical activity is associated with depression, which may, in turn, negatively impact antiretroviral therapy (ART) adherence among HIV-infected individuals; however, prior studies have not investigated the relationships between physical inactivity and ART non-adherence. PURPOSE: The purpose of this study was to examine the association of physical inactivity, depression, ART non-adherence, and viral load in HIV-infected men who have sex with men. METHODS: The sample (N = 860) was from a large, multicenter cohort of HIV-infected patients engaged in clinical care. RESULTS: Across time, depression mediated the relationship between physical inactivity and ART non-adherence (γ = 0.075) and the relationship between physical inactivity and viral load (γ = 0.05). ART non-adherence mediated the relationship between depression and viral load (γ = 0.002) and the relationship between physical inactivity and viral load (γ = 0.009). CONCLUSIONS: Low levels of physical activity predicted increased depression and poor ART adherence over time, which subsequently predicted higher viral load.