Several preventive interventions have demonstrated efficacy in reducing substance use. However, opportunities exist to further improve prevention approaches. The application of recent advances in developmental neuroscience can inform the design, implementation, and evaluation of substance use prevention programs. This paper first briefly describes the developmental integration of the prefrontal cortex with emotion and motivation centers of the brain, and the implications of this process for substance use vulnerability. Discussed next are specific examples of how developmental neuroscience can inform prevention timing, development, and evaluation. Contextual considerations are then suggested including a critical role for schools in substance misuse prevention. Finally, current theoretical and methodological challenges to the translation of developmental neuroscience to substance use prevention are discussed.