Research addressing the linkages between acculturation and markers of adolescent well-being across multiple ethnic minority groups is limited in scope and breadth, even though children of immigrant origin are the fastest growing population. We examined cross-sectional relationships between acculturation and substance use, socioemotional well-being, and academic achievement. Somali, Latino, and Hmong adolescents in Minnesota provided data as part of the EAT 2010 (Eating and Activity in Teens) cohort study ( = 1,066). Acculturation was based on nativity, language usually spoken at home, and length of residence in the United States. Chi-square, ANOVA, and regression models were used to test for differences in adolescent well-being by acculturation and ethnic group, and interaction terms were added to models to test effect modification by ethnicity. Hmong adolescents had the highest mean acculturation scores (4.4 ± 1.5), whereas Somali adolescents (2.2 ± 1.8) were the least acculturated. Independent of ethnicity, acculturation was positively associated with marijuana (OR: 1.38; CI [1.25, 1.53]) and alcohol use (OR: 1.12; CI [1.02, 1.22]), and was negatively associated with academic achievement, based on grade point average (β = -0.07; CI [-0.12, -0.03]). Interaction effects indicated significant differences by ethnicity only for academic achievement; significant associations between acculturation and academic achievement were evident only for Somali and Latino youth. Prevention programming should include supports for multilingual and multicultural learners and account for cultural assets within immigrant origin families that maintain and nurture protective factors as adolescents acculturate and transition into young adulthood. (PsycInfo Database Record (c) 2021 APA, all rights reserved).