To investigate whether long-term, chronic treatment with the phosphodiesterase-5 inhibitor vardenafil affects adrenal and testicular steroidogenesis in diabetic men, using liquid chromatography-tandem mass spectrometry. A longitudinal, prospective, investigator-started, randomized, placebo-controlled, double-blind, clinical-trial was carried out, enrolling 54 male patients affected by type 2 diabetes mellitus diagnosed within the last 5 years. In total, 26 and 28 patients were followed for 1 year and assigned to the study and placebo group, respectively. Progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, corticosterone, 11-deoxycortisol and cortisol, were evaluated using liquid chromatography-tandem mass spectrometry. No differences were seen in sex testicular steroids between study and control group. As for the adrenal gland, steroids were considered according to the zona in which they are produced. No significant differences were seen in steroid produced in zona fasciculata. For the zona reticularis, dehydroepiandrosterone significantly decreased during treatment only in the study group (p = 0.007), with higher levels at visit 2 and 8 than other visits. The dehydroepiandrosterone sulfate/dehydroepiandrosterone ratio significantly increased during treatment only in the verum group. Considering the adrenal zona glomerulosa, corticosterone significantly changed among visits both in both groups (p < 0.001), with higher levels at visit 2 (p = 0.028), 8 (p = 0.003), and 10 (p = 0.044), i.e., in coincidence with the complete clinical and instrumental examination performed only at these visits according to the study protocol. Chronically administered vardenafil reduces dehydroepiandrosterone levels and increases dehydroepiandrosterone sulfate/dehydroepiandrosterone ratio as possible consequences of modulation of steroidogenic enzymes by tissue changes in cyclic adenosine monophosphate and cyclic guanosine monophosphate availability. A possibly stress-related increase in corticosterone is suggested for the first time.