Gut microbiota respond to host physiological phenomena, yet little is known regarding shifts in the gut microbiome due to menopausal hormonal and metabolic changes in women. HIV infection impacts menopause and may also cause gut dysbiosis. We therefore sought to determine the association between menopausal status and gut microbiome composition in women with and without HIV. Gut microbiome composition was assessed in stool from 432 women (99 premenopausal HIV+, 71 premenopausal HIV-, 182 postmenopausal HIV+, 80 postmenopausal HIV-) via 16S rRNA gene sequencing. We examined cross-sectional associations of menopause with gut microbiota overall diversity and composition, and taxon and inferred metagenomic pathway abundance. Models were stratified by HIV serostatus and adjusted for age, HIV-related variables, and other potential confounders. Menopause, ie post- versus premenopausal status, was associated with overall microbial composition only in women with HIV (permutational MANOVA of Jensen Shannon Divergence: P = 0.01). In women with HIV, menopause was associated with enrichment of gram-negative order Enterobacteriales, depletion of highly abundant taxa within Prevotella copri, and alterations in other low-abundance taxa. Additionally, menopause in women with HIV was associated with enrichment of metagenomic pathways related to Enterobacteriales, including degradation of amino acids and phenolic compounds, biosynthesis of enterobactin, and energy metabolism pathways. Menopause-related differences in some low-abundance taxa were also observed in women without HIV. A changing gut microbiome may be an overlooked phenomenon of reproductive aging in women with HIV. Longitudinal assessments across all reproductive stages are necessary to confirm these findings and identify health implications.