Aim: Childhood overweight and obesity is unacceptably high in industrialised nations. School-based interventions have largely been atheoretical and ineffective. This study aimed to test a new theory-based protocol for its effects on measures of fitness and body mass index (BMI), and its proposed psychosocial mediators, during elementary after-school (out-of-school hours) care. Methods: Nine- to 12-year-old participants of YMCA-based after-school care in the southeastern Unites States were randomised into either the experimental Youth Fit 4 Life (YF4L) treatment group (n = 86) or a typical care group (n = 55) for 45 min/day. YF4L is based on social cognitive theory, emphasising mastery over physical activities and the development of self-management/self-regulatory skills to support healthy behaviours. Physiological and psychosocial variables were assessed over a 9-month elementary school year. Results: Of the overall sample, 28% were overweight or obese at baseline. YF4L was associated with significantly greater improvements in BMI, and measures of self-regulation, mood, self-efficacy, cardiovascular endurance and strength over both 3 and 9 months. Changes in self-regulation, mood and self-efficacy significantly mediated the treatment type–BMI relationship over both 3 months (R 2 = 0.12, P = 0.002) and 9 months (R 2 = 0.13, P = 0.001), with change in self-regulation being a significant independent mediator. Changes in BMI and self-regulation reciprocally reinforced one another. Gender was not a significant moderator of those relationships. Conclusions: The YF4L treatment mitigated rise in BMI in 9- to 12-year-old enrollees of after-school care. The treatment's basis in social cognitive theory was supported. Because of its positive effects and ability to utilise existing staff, large-scale application is warranted after sufficient replication.