Plasma testosterone levels have been found to decrease in older insulin-resistant male patients. Both lower total testosterone levels and a higher incidence of metabolic syndrome have also been reported. The aim of this study was to investigate the effects of high-fructose diet-induced diabetes on both the testosterone release by Leydig cells and the activity of the hypothalamus–pituitary–gonadal (HPG) axis in male rats. Male rats were fed with either standard chow (control group) or a high-fructose diet (fructose-fed group) for 21 weeks. Hyperglycemia, hyperinsulinemia, and hypertension were observed in the fructose-fed group. Moreover, plasma testosterone and LH levels decreased in the fructose-fed group compared to the control group. Sperm motility was also reduced by 15% in the fructose-fed rats. In contrast, the basal release of testosterone from rat Leydig cells was not altered by fructose feeding. Moreover, in vitro studies showed that the testosterone release, in response to different stimulants, including forskolin (an adenylyl cyclase activator, 10−5 M), 8-Br-cAMP (a permeable analog of cAMP, 10−5 M), A23187 (a calcium ionophore, 10−5 M), or 25-hydroxy-cholesterol (water-soluble cholesterol, 10−5 M), did not significantly differ between the fructose-fed and control groups. Interestingly, the release of testosterone in response to human chorionic gonadotropin (hCG, 0.05 IU/mL) was enhanced by eightfold in the control group, but elevenfold in the fructose-fed group. LH receptor expression in rat Leydig cells was also increased. Moreover, LH secretion from the anterior pituitary was altered in the fructose diet-fed group. These results suggest that fructose diet-fed rats have lower plasma testosterone levels, which can lead to a higher sensitivity of hCG in Leydig cells.