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 ( # NCT00512707 ) initially administered an optimized dose of sildenafil to 140 men, aged 40-70 years with erectile dysfunction, low serum total testosterone (< nmol/L; 330 ng/dL) and/or free testosterone (<173 pmol/L; 50 pg/mL) over 3-7 weeks. 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 nmol/L (103 ng/dL; p < ) and 110 pmol/L ( pg/mL; p < ) in total and free testosterone levels respectively. This was accompanied by parallel increases in serum DHT ( nmol/L; ng/dL; p < ) and oestradiol (14 pmol/L; pg/mL; p < ) and significant suppression of luteinizing hormone (change - units/L; p = ) levels, suggesting a direct effect at the testicular level. Androstenedione and oestrone increased by nmol/L (38 ng/dL; p = ) and pmol/L ( pg/mL; p = ), 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.