Team sport participation contributes to positive outcomes, including increased physical activity, better mental health, and enhanced social engagement. However, longitudinal studies show that team sport participation during adolescence is also associated with unhealthy lifestyle habits, including harmful substance use behaviors. Our objectives were to examine these associations in an adolescent sample, assess differences between sexes, examine the association by sport type, and investigate whether associations carry into young adulthood. Over five years of high school participants reported team sport participation, smoking status, and frequency of alcohol use, three years post-high school participants reported the same behaviors as well as marijuana use. Regression analyses accounting for individual clustering revealed that participation in team sport during high school was associated with an increased likelihood of current smoking in males and more frequent drinking in both sexes during adolescence. These relationships were strongest in individuals who participated in football. Further analyses revealed that team sport participation in high school was associated with an increased likelihood of more frequent binge drinking in young adulthood; however, team sport participation in young adulthood was associated with a decrease in the likelihood of cigarette smoking and marijuana use in young adulthood. Overall, results confirm that team sport participation in adolescence is positively associated with substance use in adolescence, and this association differs by sex. However, team sport participation in young adulthood is negatively associated with harmful substance use. Further research is needed to understand if these trends generalize beyond White adolescents and young adults.