T-2 toxin, which is produced in grain and grain products as a secondary metabolite by Fusarium species, is also potentially dangerous for human health. Up to date, no study was reported the cytotoxicity of T-2 toxin on SerW3 cells in the perspective of junctional barriers. This study focused on revealing the cytotoxic effects of T-2 on Sertoli cells associated with cell junctional barriers. In the present study, SerW3 cells were exposed to T-2 toxin at 12, 120 and 1200 ng/ml doses for 24 and 48 h. Cytotoxicity tests including cell viability (MTT), lactate dehydrogenase (LDH) cytotoxicity test and trypan blue exclusion assay were performed. Occludin, ZO-1, N-cadherin and beta-catenin were immunolabelled, expressions of occludin and N-cadherin were determined by western blotting. SerW3 cell barrier integrity was measured by transepithelial electrical resistance (TEER). Cytotoxicity caused by T-2 toxin increased in a dose dependent manner, expressions of proteins and TEER measurement decreased. This study may underlie the early targets of T-2 toxin on SerW3 cells mimicking blood testis barrier in vitro.