Objective: To investigate the intervention effect of orienteering exercises on the spatial memory ability of college students of different genders and its underlying mechanism. Methods: Forty-eight college students were randomly screened into experimental and control groups, 12 each of male and female, by SBSOD scale. The effects of 12 weeks of orienteering exercises on the behavioral performance and brain activation patterns during the spatial memory tasks of college students of different genders were explored by behavioral tests and the fNIRS technique. Results: After the orienteering exercise intervention in the experimental group, the male students had significantly greater correct rates and significantly lower reaction times than the female students; left and right dorsolateral prefrontal activation was significantly reduced in the experimental group, and the male students had a significantly greater reduction in the left dorsolateral prefrontal than the female students. The degree of activation in the left and right dorsolateral prefrontals of the male students and the right dorsolateral prefrontals of the female students correlated significantly with behavioral performance, and the functional coupling between the brain regions showed an enhanced performance. Discussion: Orienteering exercises improve the spatial memory ability of college students, more significantly in male students. The degree of activation of different brain regions correlated with behavioral performance and showed some gender differences.