In the present study, we aimed to explore the effect of environmental endocrine disruptors (EEDs) on sexual differentiation in androgen receptor (AR)-/-, AR+/- and AR+/+ male mice. By using a Cre-loxP conditional knockout strategy, we generated AR knockout mice. By mating flox-AR female mice with AR-Cre male mice, the offspring male mice which were produced were examined. Mice not subjected to any type of intervention were used as the controls. Furthermore, male mice of different genotypes were selected and further divided into subgroups as follows: the control group, bisphenol A (BPA) group and the dibutyl phthalate [corrected] (DBP) group. The expression of the Wilms tumor 1 (WT1), lutropin/choriogonadotropin receptor (LHR), 17-β-hydroxysteroid dehydrogenase type 3 (17βHSD3) and steroid-5-alpha-reductase, alpha polypeptide 2 (SRD5A2) genes was determined by RT-qPCR and western blot analysis. There was no statistically significant difference in the weight of the mice between the control group and the knockout group (P>0.05). The results revealed that, compared with the control group, in the knockout group, anogenital distance was shortened, and testicular weight and testosterone levels were decreased; estradiol levels were elevated; the differences were statistically significant (P<0.05). In the group of AR+/- male mice exposed to 100 mg/l EEDs, hypospadias was successfully induced, suggesting that EEDs are involved in the embryonic stage of sexual development in male mice. The quantitative detection of WT1, LHR, 17βHSD3 and SRD5A2 gene expression by RT-qPCR and western blot analysis indicated that these genes were significantly downregulated in the mice in the BPA group. In conclusion, exposure to EEDs induces hypospadias in heterozygous and wild-type male mice offspring during sexual differentiation, but has no effect on homozygous offspring. Therefore, EEDs play an important role during the third stage of sexual differentiation.