There is growing concern about adverse effects of bisphenol A alternatives including bisphenol B (BPB) due to their estrogenic activity. However, limited data are available concerning the influences of BPB on male reproductive development in vertebrates, especially in amphibians, which are believed to be susceptible to estrogenic chemicals. The present study investigated the effects of 10, 100 and 1000 nM BPB (2.42, 24.2 and 242 μg/L) on testis development in Xenopus laevis, a model amphibian species for studying gonadal feminization. We found that exposure to BPB from stages 45/46 to 52 resulted in down-regulation of testis-biased gene expression and up-regulation of ovary-biased gene and vitellogenin (vtgb1) expression in gonad-mesonephros complexes (GMCs) of tadpoles at stage 52, coupled with suppressed cell proliferation in testes and reduced gonadal metameres, resembling the effects of 17ß-estradiol. Moreover, an estrogen receptor (ER) antagonist ICI 182780 antagonized BPB-caused up-regulation of ovary-biased gene and vtgb1 expression to some degree, indicating that the effects of BPB on X. laevis testis differentiation could be partly mediated by ER. All observations demonstrate that early exposure to BPB inhibited testis differentiation and exerted certain feminizing effects during gonadal differentiation. When exposure was extended to post-metamorphosis, testes exhibited histological and morphological abnormalities including segmented, discontinuous and fragmented shapes, besides altered sex-dimorphic gene expression. Notably, most of BPB-caused alterations were not concentration-dependent, but the lowest concentration indeed exerted significant effects. Overall, our study for the first time reveals that low concentrations of BPB can disrupt testis differentiation partly due to its estrogenic activity and subsequently cause testicular dysgenesis after metamorphosis, highlighting its reproductive risk to amphibians and other vertebrates including humans. Our finding also implies that estrogenic chemicals-caused testis differentiation inhibition at tadpole stages could predict later testicular dysgenesis after metamorphosis, meaning a possibility of early detection of abnormal testis development caused by estrogenic chemicals.