In this study, we tested the effects of cooperative learning on students' prosocial behavior. Cooperative learning is a small-group instructional technique that establishes positive interdependence among students and, unlike most current school-based programs, does not mandate a formal curriculum. Given the emphasis in cooperative learning on peer reinforcement for positive and helpful behavior during learning activities, we hypothesized that cooperative learning would promote higher levels of prosocial behavior, and that these effects would be mediated by peer relatedness. Using a sample of 1,890 students (47.1% female, 75.2% White) from a cluster randomized trial of 15 middle schools, we found that cooperative learning significantly enhanced prosocial behavior across two years. Mediation was only partial, however, suggesting that additional mechanisms were at work, such as changes to social norms or teacher behavior. Given that cooperative learning has been shown to enhance student engagement and academic achievement in prior research, we argue that cooperative learning should be a central component of teacher training and professional development.