Abstract Background Gut dysbiosis has been linked to frailty, but its association with early mobility decline is unclear. Methods First, we determined the cross-sectional associations between walking speed and the gut microbiome in 740 older men (84 ± 4y) from the MrOS cohort with available stool samples and 400m walking speed measured in 2014–16. Then, we analyzed the retrospective longitudinal associations between changes in 6m walking speed (from 2005-06 to 2014-16, calculated by simple linear equation) and gut microbiome composition among participants with available data (702/740). We determined gut microbiome composition by 16S sequencing and examined diversity, taxa abundance, and performed network analysis to identify differences in the gut microbiome network of fast vs. slow walkers. Results Faster 400m walking speed (m/s) was associated with greater microbiome α-diversity (R=0.11; P=0.004). The association between a slower decline in 6m walking speed and higher α-diversity (R=0.07; P=0.054) approached borderline significance. Faster walking speed and less decline in walking speed were associated with higher abundance of genus-level bacteria that produce short-chain fatty acids, and possess anti-inflammatory properties, including Paraprevotella, Fusicatenibacter, and Alistipes, after adjusting for potential covariates (P<0.05). The gut microbiome networks of participants in the first vs. last quartile of walking speed (≤0.9 vs. ≥1.2 m/s) exhibited distinct characteristics, including different centrality measures (P<0.05). Conclusions Our findings suggest a possible relationship between gut microbiome diversity and mobility function, as indicated by the associations between faster walking speed and less decline in walking speed over ten years with higher gut microbiome diversity in older men.