Purpose: Girls on the Run is an after-school physical activity-based positive youth development program designed to enhance girls’ social, psychological, and physical development. We evaluated the effectiveness of the program by employing a longitudinal design and mixed methods. Methods: Girls (N = 203; aged 8–11 y) completed survey measures of positive youth development constructs (competence, confidence, connection, character, and caring), physical activity, and sedentary behavior prior to, at the end of, and 3 months after the season. Subsamples of girls, coaches, caregivers, and school personnel participated in focus groups. Coaches completed information about their team’s community impact project and number of girls who completed the season-ending 5k. Results: The full sample improved in confidence and connection, whereas girls who started below the preseason average showed the greatest gains from preseason to postseason on all measures, and scores were maintained or continued to improve at follow-up. All stakeholders in focus groups corroborated evidence of season-long improvement in social and emotional behaviors and health outcomes. Involvement in the community impact project contributed to girls’ growth in character and empathy skills. Conclusion: Findings provide empirical evidence that Girls on the Run is effective in promoting positive youth development, including season-long and lasting change in competence, confidence, connection, character, caring, and physical activity, especially among girls who exhibited lower preseason scores than their peers.