This study investigates whether family structure and parental closeness are associated with adolescent substance use and whether the association between parental closeness and adolescent substance use differs by family structure. A national representative sample of 1488 9th graders (mean age 15.8 years; SD = 0.52) was analyzed separately by gender using t-tests and a multiple group comparison in linear equation models. Adolescents from single-parent families showed a higher level of substance use compared to those living with both parents. The results also confirm the protective effect of parental closeness on adolescent substance use, though this was more effective for girls in single-parent families but less effective for boys. Therefore, boys from single-parent families in particular should be targeted by prevention programs.