Nonylphenol (NP) is the final metabolite of nonyphenol polyethoxylate (NPE), a non-ionic surfactant which is frequently incorporated into detergent and pesticide formulation. This metabolite has a structure mimicking 17β-estradiol and has been reported to have xenoestrogenic effects, to affect the endocrine system. However, its effects on testosterone release are still obscure. In the present study, we investigated the influence of NP by oral gavage or delivering via spray on testosterone release in vivo. Rat Leydig cells were prepared by enzymatic dispersion and then percoll density gradient centrifugation. Testosterone levels in the media were measured by radioimmunoassay. The data showed that oral administration of NP (100μg/kg) once per day for 30 days inhibited steroidogenesis of testosterone in rat Leydig cells. On the contrary, administration of NP via spray did not affect steroidogenesis of testosterone. In the in vivo experiments, administration of NP by oral ingestion decreased plasma testosterone concentrations in response to human chorionic gonadotropin (hCG, 5 IU/ml). Furthermore, oral NP induced a significant inhibition on the release of testosterone in vitro in response to hCG, 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), and forskolin. Moreover, administration of NP also caused a decrease on testosterone release in response to androstenedione. The in vitro experiments revealed that oral NP at a dose of 100μg/kg/day suppressed testosterone biosynthesis by inhibiting the activity of 17β-hydroxysteroid dehydrogenase (17β-HSD) and cyclic AMP pathway in Leydig cells. These results suggested that oral administration of NP inhibited steroidogenesis of testosterone in rat Leydig cells, at least in part, due to an inhibitors effect on the cAMP pathway and 17β-HSD activity.