Objective It is well known that diethylstilbestrol (DES ) can result in testicular oxidative injury, and one of its mechanisms of action is leading to dysfunction of steroidogenesis. The aim of this study was to investigate the relationship between testicular oxidative injury caused by DES and the key synthetase activities for the synthesis pathway of steroidogenesis and the possible mechanism. Methods Twenty-four 4-wk-old male Wistar albino rats were randomly divided into 4 groups, 6 rats each. Three doses of DES (0.1, 1.0 and 10 μg/kg·d) groups and a vehicle (corn oil) control group, were respectively administered by subcutaneous injection once a day for eight weeks. The rats were sacrificed after 8 weeks treatment and the body weight, testis, epididymis, prostate were weighed, respectively. The testicular tissues were homogenized and the oxidation of MDA and ROS, the activity changes of antioxidases SOD, CAT and GPx, as well as the activities of steroid synthetases 3β-HSD1 and 17β-HSD3 were determined by biochemical measurement. The levels of testosterone and LH in peripheral blood were measured by radioimmunoassay. The intensities of expression of StAR, P450scc, 3β-HSD1, 17β-HSD3-mRNA were detected by PCR. Results In the 10.0 μg/kg dose group, the weights and organ coefficients of testis and prostate were decreased significantly, the oxidation of MDA and ROS was increased distinctly and the activities of SOD, CAT, GPx, 3β-HSD1 and 17β-HSD3 were reduced. The concentration of serum testosterone was decreased in the 10.0 μg/kg dose group. In the 10.0 μg/kg and 1.0 μg/kg dose groups, the decline of LH level presented a dose-dependent manner, and the intensities of immunochemical positive staining for StAR, P450scc, 3β-HSD1 and 17β-HSD3 mRNA were decreased. Conclusions DES exposure results in disturbance of the oxidant/antioxidant balance and decline of testosterone level that induces reproductive impairment in male rats. DES induces reductions of both GPx and 3β-HSD activities which cause the decrease of testosterone synthesis. The expression of P450scc and 3β-HSD-mRNA, which are the key synthetases in biosynthetic pathway of steroidogenesis, are inhibited by DES, and it is speculated that the disturbance of steroidogenic synthesis enzymes may be one of the mechanisms of toxic effects of DES.