Regular physical activity is associated with a reduced risk of all-cause mortality, and mortality due to cardiovascular disease and cancer. Among adolescents, physical activity is associated with benefits in the prevention and control of emotional distress, and improvement of self-esteem. Countries in transitional epidemiological scenarios, such as Chile, need to develop effective strategies to improve physical activity as a way to face the epidemic of chronic diseases. The objective of this study was to evaluate the effects of a school-based physical activity program on physical fitness and mental health status of adolescents living in a low socioeconomic status area in Santiago, Chile. A quasi-experimental design was used to evaluate the effects of the program over one academic year. The study included 198 students aged 15 years old. Two ninth grade classes were randomly selected as the intervention group, with two classes of the same grade as controls. A social planning approach was used to develop the intervention. The program was designed and implemented based on student preferences, teachers' expertise and local resources. Changes in physiological and mental health status were assessed. After the intervention, maximum oxygen capacity achieved a significant increase of 8.5% in the intervention versus 1.8% in the control group (p < 0.0001). Speed and jump performance scores improved significantly more in the intervention versus the control group (p > 0.01). Anxiety score decreased 13.7% in the intervention group versus 2.8% in the control group (p < 0.01), and self-esteem score increased 2.3% in the intervention group and decreased 0.1% in the control group after the end of the program (p < 0.0001). No significant change was observed in the depressive score. Student participation and compliance with the program was > 80%. To conclude, a school-based program to improve physical activity in adolescents of low socioeconomic status, obtained a high level of participation and achieved significant benefits in terms of physical fitness and mental health status.