Background: Gender makes a difference in health and physical activity. This research aimed to identify the gender difference in the relationship of PA and subjective happiness among Chinese university students.Methods: A cross-sectional survey was conducted in Shanghai Jiao Tong University from July 7 to 17 in 2021, using an anonymous online self-report questionnaire. The questionnaire included the Chinese version of the International Physical Activity Questionnaire-Short Form, the Subjective Happiness Scale, the Depression Anxiety Stress Scale-21. The demographic and health-related factors were (...) also reported. Linear regression was carried out to identify the relationship of PA and subjective happiness.Findings: In total, 1,512 students from three comprehensive schools completed the survey. The SHS score was 17.20 ± 3.44, and it was 17.00 ± 3.23 in males, and 17.75 ± 3.93 in females, respectively. There was no gender difference in vigorous, moderate PA, or walk min/week, while female students had longer sedentary behavior hours/day than males. Male students scored higher in each subscale of DASS-21. After controlling for confounders, vigorous PA min/week and sedentary behavior hours/day were significantly positively associated with happiness in male students. In contrast, walk min/week was significantly positively associated with happiness in female students.Interpretation: This research demonstrated a significant gender difference in the association between PA and happiness. Policymakers and college management should pay more attention to PA programs to promote students’ happiness and mental health status. (shrink)