OBJECTIVE: Men are currently underrepresented in weight loss trials despite similar obesity rates, which limit our understanding about the most effective elements of treatment for men. The purpose of this study was to test the theoretical (autonomous motivation, self-efficacy, outcome expectancies, and self-regulation) and behavioral (calorie intake, physical activity, self-weighing) mediators of a men-only, Internet-delivered weight loss intervention focused on innovative and tailored treatment elements specifically for men. METHOD: Data comes from a 6-month randomized trial (N = 107) testing the intervention compared to a waitlist control group. Changes in the theoretical mediators between baseline and 3 months were tested as mediators of the intervention effect on weight change at 6 months in both single and multiple mediator models. Changes in behaviors between baseline and 6 months were tested in the same manner. RESULTS: The intervention produced greater weight losses compared to the control group (-5.57 kg +/- 6.6 vs. -0.65 kg +/- 3.3, p < 0.001) and significant changes (p's < 0.05) in most of the theoretical and behavior mediators. In multiple mediator models, changes in diet-related autonomous motivation, self-efficacy, and self-regulation all significantly mediated the relationship between the intervention and weight loss. The intervention effect was also mediated by changes in dietary intake and self-weighing frequency. CONCLUSIONS: By testing the theoretical mediators of this intervention in a multiple mediator context, this study contributes to current knowledge related to the development of weight loss interventions for men and suggests that interventions should target diet-focused constructs.