Background Adverse childhood experiences are prevalent robust risk factors for the development of substance use problems. However, less is known about the causal mechanisms that explain these relationships. While directly preventing adverse childhood experiences is ideal, it is not always possible. In such cases, the mechanisms themselves may be amenable to intervention, allowing for the effective prevention of problematic substance use among children exposed to adversity. Identifying such mechanisms is therefore a critical step for efforts aiming to reduce the high individual and societal burdens associated with substance use globally. Objective This study aims to systematically identify and synthesize evidence on the modifiable mediators and moderators of the relationship between adverse childhood experiences and substance use outcomes in young people (age 10-24 years). Methods A systematic review will be conducted using PubMed, MEDLINE, PsycINFO, Web of Science, and CINAHL databases to determine the modifiable mediators and moderators of the relationship between adverse childhood experiences and substance use in young people. Data from the review will be qualitatively synthesized, unless we identify a sufficient number of studies (at least five) that examine the same type of adversity (eg, physical or sexual abuse) and the same mediator/moderator, in which case a quantitative synthesis (meta-analysis) will be conducted. If a quantitative synthesis is warranted, standardized effect estimates of the indirect (mediated) effect between adverse childhood experiences and substance use outcomes will be combined using a random-effects meta-analysis. Mediators/moderators will be grouped according to a socioecological perspective, using the four levels of individual, interpersonal, community, and public policy/culture. Results Electronic searches were completed in August 2019. A total of 4004 studies were included for screening after removing duplicates. After evaluating titles and abstracts against eligibility criteria, a further 3590 studies were excluded, leaving 415 studies for full-text screening. The results of the review are expected to be available by December 2020. Conclusions The mechanisms linking adverse childhood experiences and substance use outcomes in young people are vital targets for substance use prevention efforts. This review will provide evidence to inform the development of prevention strategies in order to interrupt the negative life trajectory that can begin with childhood adversity. Trial Registration PROSPERO International Prospective Register of Systematic Reviews CRD42020148773; https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020148773 International Registered Report Identifier (IRRID) DERR1-10.2196/22368