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Microbial translocation and monocyte activation predict mortality in treated HIV. We examined whether substance use independently contributes to these pathophysiologic processes.Cross-sectional study at baseline for a randomized controlled trial.HIV-positive, methamphetamine-using MSM with undetectable HIV viral load (less than 40 copies/ml) were enrolled. We examined if plasma biomarkers of monocyte activation and intestinal barrier integrity were associated with the following: reactive urine toxicology results (Tox+) for stimulants (i.e., methamphetamine or cocaine) and substance use severity measured by the Addiction Severity Index. Multiple linear regression models adjusted for age, antiretroviral therapy regimen, CD4+ T-cell count, interleukin-6, and alcohol use severity.The sample of 84 virally suppressed MSM had a median CD4+ T-cell count of 645 cells/μl. Those who were Tox+ for stimulants displayed higher soluble CD14 (sCD14) levels (2087 versus 1801 ng/ml; P = 0.009), and this difference remained significant after adjusting for covariates (standardized beta = 0.23, P = 0.026). Greater substance use severity was also independently associated with higher sCD14 after adjusting for covariates (standardized beta = 0.29, P = 0.013). Being Tox+ for stimulants and substance use severity were not associated with soluble CD163 (sCD163) or intestinal fatty acid binding protein (iFABP) levels (P > 0.05).Monocyte activation is one plausible mechanism by which stimulant use may increase clinical HIV progression.