Emerging evidence suggests that dietary fats may influence testicular function. However, most of the published literature on this field has used semen quality parameters as the only proxy for testicular function. We examined the association of fat intake with circulating reproductive hormone levels and testicular volume among healthy young Spanish men. This is a cross-sectional study among 209 healthy male volunteers conducted between October 2010 and November 2011 in Murcia Region of Spain. Participants completed questionnaires on lifestyle, diet, and smoking, and each underwent a physical examination, and provided a blood sample. Linear regression was used to examine the association between each fatty acid type and reproductive hormone levels and testicular volumes. Monounsaturated fatty acids intake was inversely associated with serum blood levels of calculated free testosterone, total testosterone, and inhibin B. A positive association was observed between the intake of polyunsaturated fatty acids, particularly of omega-6 polyunsaturated fatty acids, and luteinizing hormone concentrations. In addition, the intake of trans fatty acids was associated with lower total testosterone and calculated free testosterone concentrations (P trend = 0.01 and 0.02, respectively). The intake of omega-3 polyunsaturated fatty acids was positively related to testicular volume while the intake of omega-6 polyunsaturated fatty acids and trans fatty acids was inversely related to testicular volume. These data suggest that fat intake, and particularly intake of omega 3, omega 6, and trans fatty acids, may influence testicular function.