Bisphenol A, an estrogenic environmental toxicant, has been implicated to have hazardous effects on reproductive health in humans and rodents. However, there are conflicting reports in the literature regarding its effects on male reproductive function. In this study, it was shown that in adult rats treated with acute doses of bisphenol A, a small but statistically insignificant percentage of seminiferous tubules in the testes displayed signs of germ cell loss, consistent with some earlier reports. It also failed to disrupt the blood-testis barrier in vivo. This is possibly due to the low bioavailability of free bisphenol A in the systemic circulation. However, bisphenol A disrupted the blood-testis barrier when administered to immature 20-day-old rats, consistent with earlier reports concerning the higher susceptibility of immature rats towards bisphenol A. This observation was confirmed using primary Sertoli cells cultured in vitro with established tight junction-permeability barrier that mimicked the blood-testis barrier in vivo. The reversible disruption of Sertoli cell tight junction barrier by bisphenol A was associated with an activation of ERK, and a decline in the levels of selected proteins at the tight junction, basal ectoplasmic specialization, and gap junction at the blood-testis barrier. Studies by dual-labeled immunofluorescence analysis and biotinylation techniques also illustrated declining levels of occludin, connexin 43, and N-cadherin at the cell-cell interface following bisphenol A treatment. In summary, bisphenol A reversibly perturbs the integrity of the blood-testis barrier in Sertoli cells in vitro, which can also serve as a suitable model for studying the dynamics of the blood-testis barrier.