We examined associations between stimulant use (methamphetamine and cocaine) and other substances (nicotine, marijuana, alcohol and inhaled nitrites) with immune function biomarkers among HIV-seropositive (HIV +) men taking highly active antiretroviral therapy (ART) and HIV-seronegative (HIV-) men in the Multicenter AIDS Cohort Study. Among HIV + men, cumulative adherence to ART (4.07, 95% confidence interval [CI]: 3.52, 4.71, per 10 years of adherent ART use), and recent cohort enrolment (1.38; 95% CI: 1.24, 1.55) were multiplicatively associated with increase in CD4+/CD8+ ratios. Cumulative use of methamphetamine (0.93; 95% CI: 0.88, 0.98, per 10 use-years), cocaine (0.93; 95% CI: 0.89, 0.96, per 10 use-years) and cumulative medical visits (0.99; 95% CI: 0.98, 0.99, per 10 visit-years), each showed small negative associations with CD4+/CD8+ ratios. Among HIV- men, cumulative medical visits (0.996; 95% CI: 0.993, 0.999), cumulative number of male sexual partners (0.999; 95% CI: 0.998, 0.9998, per 10 partner-years) and cigarette pack-years (1.10; 95% CI: 1.02, 1.18, per 10 pack-years) were associated with CD4+/CD8+ ratios over the same period. ART adherence is associated with a positive immune function independent of stimulant use, underscoring the influence of ART on immune health for HIV+ men who engage in stimulant use.