Objective: To investigate the association between habitual physical activity levels and brain temporal lobe volumes, and the interaction with the brain-derived neurotrophic factor (BDNF) Val66- Met polymorphism. Methods: This study is a cross-sectional analysis of 114 cognitively healthy men and women aged 60 years and older. Brain volumes quantified by MRI were correlated with self-reported physical activity levels. The effect of the interaction between physical activity and the BDNF Val66Met polymorphism on brain structure volumes was assessed. Post hoc analyses were completed to evaluate the influence of the APOE e4 allele on any found associations. Results: The BDNF Val66Met polymorphism interacted with physical activity to be associated with hippocampal (b 5 20.22, p 5 0.02) and temporal lobe (b 5 20.28, p 5 0.003) volumes. In Val/Val homozygotes, higher levels of physical activity were associated with larger hippocampal and temporal lobe volumes, whereas in Met carriers, higher levels of physical activity were associated with smaller temporal lobe volume. Conclusion: The findings from this study support higher physical activity levels in the potential attenuation of age- And disease-related hippocampal and temporal lobe volume loss in Val/Val homozygotes.