Phosphodiesterase-5-inhibitors, such as sildenafil, increase intracavernosal cyclic guanosine monophosphate levels, which results in corporal smooth muscle relaxation and penile erection. Here, we determined the effects of sildenafil administration on the hypothalamic-pituitary-gonadal axis in men with erectile dysfunction and low testosterone levels. The Testosterone and Erectile Dysfunction trial (ClinicalTrials.gov # NCT00512707) initially administered an optimized dose of sildenafil to 140 men, aged 40-70years with erectile dysfunction, low serum total testosterone (<11.4nmol/L; 330ng/dL) and/or free testosterone (<173pmol/L; 50pg/mL) over 3-7weeks. Sex steroids and gonadotropins were measured at baseline and after sildenafil optimization in a longitudinal study without a separate control group. Serum testosterone, dihydrotestosterone (DHT) and oestrogens were measured using liquid chromatography-tandem mass spectrometry. Administration of an optimized dose of sildenafil was associated with mean increases of 3.6nmol/L (103ng/dL; p<0.001) and 110pmol/L (31.7pg/mL; p<0.001) in total and free testosterone levels respectively. This was accompanied by parallel increases in serum DHT (0.17nmol/L; 4.9ng/dL; p<0.001) and oestradiol (14pmol/L; 3.7pg/mL; p<0.001) and significant suppression of luteinizing hormone (change -1.3units/L; p=0.003) levels, suggesting a direct effect at the testicular level. Androstenedione and oestrone increased by 1.3nmol/L (38ng/dL; p=0.011) and 10.7pmol/L (2.9pg/mL; p=0.012), respectively, supporting a possible effect of sildenafil on adrenal steroidogenesis. In conclusion, sildenafil administration was associated with increased testosterone levels likely ascribable to a direct effect on the testis.