Bisphenol S (BPS) is a novel bisphenol A (BPA) analogue, a ubiquitous environmental pollutant that disrupts male reproductive system. Whether BPS affects Leydig cell maturation in male puberty remains unclear. Male Sprague-Dawley rats (age of 35 days) were daily gavaged to 0, 1, 10, 100, and 200 mg/kg/day from postnatal days 35-56. BPS at 1-10 mg/kg/day and higher doses markedly reduced serum testosterone and progesterone levels but it at 200 mg/kg/day significantly increased estradiol level. BPS at 100 and 200 mg/kg/day significantly elevated serum luteinizing hormone (LH) levels. BPS at 1-10 mg/kg/day and higher doses significantly reduced inhibin A and inhibin B levels. BPS at 100 and 200 mg/kg/day markedly increased CYP11A1+ Leydig cell number, but did not affect HSD11B1+ (a mature Leydig cell marker) cell number. BPS at 10 mg/kg/day and higher doses significantly downregulated the expression of Cyp11a1 and at 100 and 200 mg/kg/d significantly lowered Cyp17a1, Hsd11b1, and Nr5a1 in the testes. BPS at 100 and/or 200 mg/kg/day significantly elevated Lhb in the pituitary. BPS at 100 and 200 mg/kg/day significantly increased the phosphorylation of AKT1, AKT2, and CREB without affecting total AKT1, AKT2, and CREB levels. BPS at 1-100 μM significantly suppressed testosterone production and induced proliferation of primary immature Leydig cells after 24 h of treatment and these actions were reversed by estrogen receptor α antagonist, ICI 182780, and partially reversed by vitamin E. BPS at 0.1-10 μM significantly increased oxidative stress of Leydig cells in vitro. BPS also directly inhibited 17β-hydroxysteroid dehydrogenase 3 activity at 10-100 μM. In conclusion, BPS causes hypergonadotropic androgen deficiency in male rats during pubertal exposure via activating ESR1 and inducing ROS in immature Leydig cells and directly inhibiting 17β-hydroxysteroid dehydrogenase 3 activity.