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Background: Given its ability to exacerbate health inequities through its disproportionate impact on low-income communities, the need exists to better understand factors that influence substance use among adolescents. Moreover, given its multi-etiological nature, preventing adolescent substance use requires addressing intrapersonal, interpersonal, and environmental behavioral influences. Using data from a school-based program that was implemented among youth in low-income Chicago public schools, we evaluated the mechanisms by which the program influenced youth substance use. Method: Data for this study come from the longitudinal, Chicago randomized control trial of Positive Action (PA), a social-emotional and character development program. Fourteen diverse, low-performing, high-poverty schools were matched into 7 pairs, and schools within each pair were randomized to the treatment (receipt of PA) or control condition. A diverse, dynamic cohort of approximately 1,200 students were followed starting in grade 3 up until grade 8. We used longitudinal mediation analysis and an SEM framework to test the overall effect of the PA program on substance use, and to test the mediational pathways for one intrapersonal (i.e., self-control), one interpersonal (i.e., deviant peer affiliation), and one environmental (i.e., school attachment) risk factor. Results: For all students, when examining the slope of each mediator as a predictor of each substance use outcome, greater self-control was associated with lower levels of alcohol, cigarette and marijuana use, greater deviant peer affiliation was associated with greater levels of alcohol, cigarette and marijuana use, and greater school attachment was marginally associated with lower levels of alcohol use. Participants who received the PA program, as compared to students in control schools, were less likely to engage in drinking-related behaviors at grade 8. Results from longitudinal mediation analyses suggest the impact of PA on alcohol, cigarette and marijuana use is mediated by reductions in deviant peer affiliation. Discussion: Our results demonstrate that a school-wide social-emotional program was able to reduce substance use and suggest that the most influential mediational pathway by which the program achieved this impact is through improvements in affiliation with peers who engage in high-risk behaviors. This aligns with developmental research noting the influential role of peers on adolescent behavior. Implications for prevention, particularly in high-risk settings, are discussed.