To explore the longitudinal relationship among physical activity, peer acceptance, and pro-social behavior in rural left-behind children, a physical activity questionnaire, peer acceptance scale, and pro-social behavioral tendency scale were used to conduct three follow-up surveys (T1, T2, and T3) over a period of six months with 612 rural left-behind children in Ji＇an City, Jiangxi Province. The results showed that (1) there were significant concurrent and longitudinal correlations among physical activity, peer acceptance, and pro-social behavior; (2) cross-lagged analyses indicated that physical activity and pro-social behavior positively predicted each other; (3) longitudinal mediation analyses found that T1 physical activity positively predicted T3 pro-social behavior via T2 peer acceptance, and T1 pro-social behavior predicted T3 physical activity through T2 peer acceptance; (4) peer acceptance played a longitudinal mediating role between physical activity and pro-social behavior. These findings suggest that physical activity and peer acceptance facilitate the development of pro-social behaviors in rural left-behind children and can serve as effective interventions to promote their psychological health and social adaptation.