Objective To explore the relationship between physical activity and prosocial behavior in college students, and to examine whether self-perception and gender may play mediating and moderating roles, respectively, in that relationship. Methods The International Physical Activity Questionnaire-long form, Prosocial Tendencies Measure, and Self-perception Scale were used to survey 647 college students in Yangzhou, China. Internal consistency testing, one-way analyses of variance (ANOVAs) across physical activity levels, exploratory factor analysis, correlation testing, mediation effect testing (independent variable, physical activity; mediating variable, self-perception; dependent variable, prosocial behavior), bootstrap testing and moderated mediation testing were conducted. Results Physical activity level was not found to be a direct predictor prosocial behavior in college students. Self-perception was found to play a mediating role between physical activity and prosocial behavior. Conclusion Physical activity is not directly predictive college students’ prosocial behavior tendencies, but it is indirectly predictive through self-perception. This study explores the relationship between the three variables and the path of the relationship, deepening the research related to the relationship between physical activity and prosocial behavior, providing ideas for fostering prosocial behavior in Chinese universities, as well as providing a theoretical basis for possible future empirical research.