<p>Aging-related declines in mobility are more common in women than men. The gut microbiome may play a role in physical function, but sex-specific roles are unknown. In adults ≥50 years old (<i>n</i> = 1187 women, 585 men), we examined associations of self-reported mobility impairment with gut microbiome assessed by stool shotgun sequencing, and examined heterogeneity by sex. Gut microbiome α-diversity was lower and overall composition (β-diversity) altered in women with mobility impairment compared to without, but this was not the case in men. Fifteen microbiome species were associated with mobility impairment in both women and men, including enrichment of <i>Streptococcus</i> and <i>Lactobacillus</i> and depletion of <i>Eubacterium</i> species. An additional 84 species were associated with mobility impairment in women only, including enrichment of Gammaproteobacteria species, but none were associated with mobility impairment in men only. Correlations of impaired mobility-related microbiome scores, derived from universal and women-specific microbiome species, with serum metabolites (<i>n</i> = 385) suggested that impaired mobility-related species may be involved in synthesis of imidazole propionate and deoxycholic acid metabolites, while species depleted with mobility impairment may be involved in sex hormone metabolism and guanidinoacetate production, the latter in women only. Gut microbiota may play a role in physical function and sex differences therein.</p>