BACKGROUND: The widespread availability of antiretroviral therapy (ART) has dramatically reduced mortality and improved life expectancy for people living with HIV (PLWH). However, even with HIV-1 suppression, chronic immune activation and elevated inflammation persist and have been linked to a pro-inflammatory gut microbiome composition and compromised intestinal barrier integrity. PLWH in urban versus rural areas of sub-Saharan Africa experience differences in environmental factors that may impact the gut microbiome and immune system, in response to ART, yet this has not previously been investigated in these groups. To address this, we measured T cell activation/exhaustion/trafficking markers, plasma inflammatory markers, and fecal microbiome composition in PLWH and healthy participants recruited from an urban clinic in the city of Harare, Zimbabwe, and a district hospital that services surrounding rural villages. PLWH were either ART naïve at baseline and sampled again after 24 weeks of first-line ART and the antibiotic cotrimoxazole or were ART-experienced at both timepoints. RESULTS: Although expected reductions in the inflammatory marker IL-6, T-cell activation, and exhaustion were observed with ART-induced viral suppression, these changes were much more pronounced in the urban versus the rural area. Gut microbiome composition was the most highly altered from healthy controls in ART experienced PLWH, and characterized by both reduced alpha diversity and altered composition. However, gut microbiome composition showed a pronounced relationship with T cell activation and exhaustion in ART-naïve PLWH, suggesting a particularly significant role for the gut microbiome in disease progression in uncontrolled infection. Elevated immune exhaustion after 24 weeks of ART did correlate with both living in the rural location and a more Prevotella-rich/Bacteroides-poor microbiome type, suggesting a potential role for rural-associated microbiome differences or their co-variates in the muted improvements in immune exhaustion in the rural area. CONCLUSION: Successful ART was less effective at reducing gut microbiome-associated inflammation and T cell activation in PLWH in rural versus urban Zimbabwe, suggesting that individuals on ART in rural areas of Zimbabwe may be more vulnerable to co-morbidity related to sustained immune dysfunction in treated infection. Video Abstract.