ImportanceSubstance use is high among American Indian/Alaska Native adolescents. Pain interference is a risk factor for substance use, and identifying potential protective factors, such as Tribal identity, is important to help inform culturally grounded substance use prevention strategies.ObjectiveTo examine the protective potential of Tribal identity as a moderator in the association between pain interference and substance use in American Indian/Alaska Native adolescents.Design, Setting, and ParticipantsThis cross-sectional study of 10th-grade students self-identifying as American Indian/Alaska Native was conducted among students enrolled at 20 high schools on or near the Cherokee Nation Reservation. Data for this study were collected from the baseline assessments of a cluster randomized trial to prevent substance use among adolescents living on or near the Cherokee Nation Reservation. Inclusion criteria for high schools’ participation included being located within counties that partially or fully fall within the Cherokee Nation reservation, a town population of 3000 individuals or fewer, and class sizes between 30 and 100 students. Exclusion criteria included high schools within metropolitan and micropolitan cores (per the US Census Bureau’s Rural-Urban Commuting Area codes) and the existence of an established community drug prevention coalition. Student surveys were conducted from September 2021 to May 2024.ExposurePain interference in the 7 days prior to baseline assessment.Main Outcomes and MeasuresThe primary outcomes were past 30-day alcohol use, marijuana use, and prescription opioid misuse. Generalized estimating equations Poisson models were used, with an exchangeable correlation structure clustered on the school level using baseline data from a cluster randomized trial to prevent alcohol and substance use among adolescents. For each substance, a separate multivariable model was fit, which included pain interference, Tribal identity, an interaction term between pain interference and Tribal identity, age, gender, food insecurity, anxiety symptoms, and depressive symptoms.ResultsAmong 514 self-identified American Indian/Alaska Native students at 20 high schools on or near the Cherokee Nation Reservation, mean (SD) participant age was 15.59 (0.62) years, and 252 participants (49.0%) self-identified as female. Pain interference was associated with alcohol use and prescription opioid misuse in American Indian/Alaska Native adolescents, controlling for age, gender, food insecurity, anxiety symptoms, and depressive symptoms. Tribal identity significantly moderated the association between pain interference and alcohol use (coefficient, −0.13; 95% CI, −0.23 to −0.02).Conclusions and RelevanceWhen Tribal identity was high, the adverse association of pain interference with alcohol use was significantly attenuated. This demonstrates a protective role of Tribal identity on alcohol use, which can inform future substance use prevention efforts among American Indian/Alaska Native adolescents.Trial RegistrationClinicalTrials.gov Identifier: NCT04839978