Background: To investigate the association between step counts and brain volumes (BVs)—global and 6 a priori selected cognition-related regions of interest—in Japanese men aged 40–79 years. Methods: The authors analyzed data from 680 cognitively intact participants of the Shiga Epidemiological Study of Subclinical Atherosclerosis—a population-based observational study. Using multivariable linear regression, the authors assessed cross-sectional associations between 7-day step counts at baseline (2006–2008) and BVs at follow-up (2012–2015) for age-stratified groups (<60 y and ≥60 y). Results: In the older adults ≥60 years, step counts at baseline (per 1000 steps) were associated with total BV at follow-up (β = 1.42, P = .022) while adjusted for potential covariates. Regions of interest-based analyses yielded an association of step counts with both prefrontal cortexes (P < .05) in older adults, while the left entorhinal cortex showed marginally significant association (P = .05). No association was observed with hippocampus, parahippocampal, cingulum, and cerebellum. No association was observed in younger adults (<60 y). Conclusions: The authors found a positive association between 7-day step counts and BVs, including prefrontal cortexes, and left entorhinal cortex in apparently healthy Japanese men.