The primary aim of this study was to test the effect of a school-based physical activity intervention on adolescents' performance in mathematics. A secondary aim was to explore potential mechanisms that might explain the intervention effect.The Activity and Motivation in Physical EDucation intervention was evaluated using a two-arm cluster randomized controlled trial in 14 secondary schools located in low socioeconomic areas of Western Sydney, Australia. Study participants (n = 1173) were grade 8 students (mean age = 12.94 yr, SD = 0.54). The multicomponent intervention was designed to help teachers maximize students' opportunities for moderate-to-vigorous physical activity (MVPA) during physical education (PE) and enhance students' motivation toward PE. Mathematics performance was assessed as part of national testing in grade 7, which was the year before the trial began and then again in grade 9. Potential mediators were: (i) proportion of PE lesson time that students spent in MVPA and leisure time MVPA (%), measured using Actigraph GT3X+ accelerometers, and (ii) students' self-reported engagement (behavioral, emotional, and cognitive) during mathematics lessons. Mediators were assessed at baseline (grade 8) and follow-up (grade 9, 14-15 months after baseline).The effect of the intervention on mathematics performance was small-to-medium (β = 0.16, P < 0.001). An intervention effect was observed for MVPA% in PE (β = 0.59, P < 0.001), but not for leisure time MVPA or any of the engagement mediators. There were no significant associations between changes in potential mediators and mathematics performance.The Activity and Motivation in Physical EDucation intervention had a significant positive effect on mathematics performance in adolescents. However, findings should be interpreted with caution as the effect was small and not associated with changes in hypothesized mediators.