Previous research has shown a decline in physical activity (PA) across college years, females being less physically active compared with males. Scholars have suggested studies to understand gender differences in PA and to examine motivational processes to facilitate college students' PA. Grounded in self-determination theory, the purpose of this study was to investigate the relationships between college students' exercise motivation and weekly PA participation. The study included 96 college students (33 males, 63 females, aged 18 to 24) in a metropolitan college. Findings confirmed a significant gender difference, with males responding more positively concerning intrinsic motivation (t = 3.40, p = 0.001). In addition, through an analysis of variance, we found level of PA had a significant interaction with intrinsic motivation, F(1, 94) = 9.45, p < 0.001, and identified regulation, F(1, 94) = 6.45, p = 0.003. Furthermore, least significant difference tests showed that the differences occurred between inactive and moderately active groups and inactive and very active groups (p values between 0.011 and 0.000). Results from this study concerning motivation for PA with this age group support the premise that self-determined motivation is strongly linked to higher PA participation. The findings of this study provide a better understanding of how to assist college-aged students to live a more physically active and healthy lifestyle.