PURPOSE: To examine and compare the role of self-assessed sport and physical activity involvement on the health-related quality of life among undergraduate student-athletes and general undergraduate college students. DESIGN: Cross-sectional survey. Data set was examined for differences in physical and mental health by self-assessed sport and physical activity level. SETTING: Large Midwestern University in the fall of 2016. PARTICIPANTS: A combined data set representing undergraduate Division I student athletes (n = 842) and general undergraduate students (n = 1322). MEASURES: Veterans RAND 12 Item Health Survey (VR-12), as measure of health-related quality of life, comprised of physical component score (PCS) and mental component score (MCS). Self-assessed sport and physical activity level categorized as Division I athlete, club athlete, intramural player, student who works out regularly, or student who is physically inactive. ANALYSIS: Standard univariable statistics described the study population. Two-sample t tests and χ tests were conducted, as appropriate, to compare Division I student-athletes to the general undergraduate group. Multivariable linear regression models were then built to assess associations between physical activity level and year in school with VR-12 outcomes, after adjusting for sex. All pairwise interactions were considered for inclusion in the final models. Adjusted least-square means were calculated for all variables in the model; pairwise comparisons were adjusted for multiple comparisons via Tukey-Kramer adjustment criteria. A linear test for trend was also conducted for the association between VR-12 MCS and increasing physical activity. RESULTS: Significant differences in MCS were noted between levels of sport and physical activity; however, such differences were not detected in PCS. After controlling for sex, a positive relationship between increased sport and physical activity level and greater MCS was found. CONCLUSIONS: This study represents the first prospective assessment of health-related quality of life among undergraduate athletes and general college students. Higher levels of sport and physical activity were associated with more positive mental health in these populations.