Background Arsenic is recognized as a kind of developmental toxicant, which can pass through the placenta barrier and induce health defects in offspring. However, the effects of environmental levels of arsenic exposure during gestation on the reproductive system of adult male offspring remain unclear. Objective To investigate the impact of environmental levels of arsenic exposure during gestation on testosterone synthesis and sperm quality in F1 adult male rats. Methods Forty sexually mature Wistar female rats were randomly divided into four groups according to body weight, namely control group, low-dose sodium arsenite (NaAsO₂) group, middle-dose NaAsO₂ group, and high-dose NaAsO₂ group. They were mated with sexually mature Wistar male rats in a ratio of 2:1, and the day with presence of a vaginal plug or spermatozoa in the vaginal smear was designated as gestational day 0 (GD0). Pregnant rats were provided drinking water containing 0, 1, 5,^, or 25 mg·L⁻¹ NaAsO₂ until delivery. At postnatal day 70, the F1 male rats were euthanized. Anogenital distance was measured, testis and epididymis were weighed, and associated organ coefficients were calculated. Epididymal sperm quality was evaluated. The histological changes of testis were observed. The levels of testosterone and estradiol in serum were determined with ELISA kit. The testicular mRNA relative expression levels of key steroidogenic enzymes were determined by quantitative real-time PCR. The protein relative expression levels of key steroidogenic enzymes were determined by Western blotting. Results Compared with the control group, the testicular coefficients and epididymis coefficients were increased in the low- and middle-dose groups (P<0.05), and the epididymis coefficient was also increased in the high-dose group (P<0.05). As for the percentage of sperm motility, compared to the control group, grade Ⅰ sperm cells were increased in the low-dose group, but significantly decreased in the middle- and high-dose groups; grade Ⅱ and Ⅲ sperm cells were decreased in the low- and high-dose groups; grade Ⅳ sperm cells were significantly increased in the middle- and high-dose groups; all the differences above were statistically significant (P<0.05). Compared with the control group, there was a significant increase in serum testosterone levels in all treated groups (P<0.05), and the serum estradiol levels were significantly decreased in the high-dose group (P<0.05). Meanwhile, compared with the control group, the relative mRNA expression levels of Hsd3β1 and Cyp19a1 were decreased (P<0.05), while those of StAR and Cyp11a1 were increased in the high-dose group (P<0.05). In addition, the relative protein expression levels of CYP11A1 were significantly increased in all treated groups compared with the control group (P<0.05). Conclusion Environmental levels of arsenic exposure during gestation can up-regulate testosterone level and reduce sperm quality, and is a potential risk for reproductive dysfunction in adult male offspring.