Increased physical activity has shown positive effects on various hippocampal memory functions through accumulating evidence that physical exercise and higher cardiorespiratory fitness can enhance human performance on nonspatial mnemonic discrimination tasks that rely on hippocampal pattern separation. However, there is less direct evidence of exercise effects on spatial pattern separation in humans, despite evidence for this association in rodent models. We examined the influence of strenuous exercise habits on spatial mnemonic discrimination among 176 young adults. We used a delayed match-/non-match-to-sample (same/different) task to assess pattern separation for spatial locations across varying degrees of similarity. Participants who reported regularly engaging in strenuous exercise three or more times per week performed significantly better than those who reported engaging in strenuous exercise fewer than three times per week, even when pattern separation tasks involved higher spatial similarity. These apparent exercise effects were observed for female, but not male, participants. These findings support likely benefits of strenuous exercise habits for human spatial pattern separation skills, and they suggest a need to explore potential interaction effects of exercise and gender.