The aim of this study was to explore the effects of social support and behavioral regulation of exercise on physical activity (PA) and quality of life (QoL), in a Portuguese school-based intervention. We hypothesized that serial mediation effects would be present leading to greater levels of PA and QoL. The sample comprised 1042 students (549 boys), aged 10-16 years, BMI = 19.31 ± 3.51, allocated to two groups of schools: control (n = 207) and intervention (n = 835). This study will report the 24 months results of the program, which aimed to develop healthy lifestyles. Questionnaires were used to measure PA, QoL, motivation to exercise and social support. There was no direct impact of the intervention on QoL or PA. Serial mediation analyses were conducted. Social support (P < 0.019) and intrinsic motivation (P = 0.085) increased more on intervention group. Indirect effects were observed in all serial mediation models. The positive indirect effects on PA and QoL were explained by the increase on peer/parent support in serial with the increase in intrinsic motivation (P < 0.01). Parental support led to an increase on external motivation (P < 0.05), which buffered the effects of the intervention. This school-based intervention promoted the development of social support and motivational mechanisms that explained higher levels of PA and QoL.