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Physical activity enhances subjective well-being, yet the underlying mechanisms remain unclear. This study examined the mediating role of emotion regulation and the moderating influence of gender roles among 896 university students (411 men). Physical activity was positively correlated with life satisfaction (r = 0.33, p < 0.01) and interpersonal relationship quality (r = 0.19, p < 0.01), and negatively correlated with depression (r = -0.23, p < 0.01). Emotion regulation partially mediated these associations. Moderated mediation analyses indicated that the indirect effects were significant only for students adhering to masculine (β = 0.30, p < 0.01) and feminine (β = 0.13, p < 0.05) orientations, but not for androgynous or undifferentiated groups. These results highlight that the psychological benefits of physical activity are not uniform but shaped by individual differences in gender-role orientations, underscoring the need for tailored strategies that match students' gender-role identities when promoting mental health.