The RhoA/ROCK signaling pathway plays a significant role in transforming growth factor (TGF)-β1-mediated epithelial-mesenchymal transition (EMT). It remains unclear, however, whether the RhoA/ROCK signaling pathway mediates TGF-β1-induced EMT by promoting the dissolution of tight junctions (TJs) in renal proximal tubular epithelial cells. In this study, we aimed to investigate the association between TGF-β1-mediated Rho/ROCK signaling and TJs in a cell line derived from human renal proximal tubular cells (HK-2 cells). HK-2 cells were treated with 5 ng/ml TGF-β1 for 0, 12, 24 and 48 h. Zona occludens protein 1 (also known as tight junction protein 1; ZO-1) and occludin mRNA and protein levels were determined by real-time PCR and western blot analysis, respectively. The HK-2 cells were then divided into three groups: a control group (serum-free culture medium for 24 h); a TGF-β1 group (treated with 5 ng/ml TGF-β1 for 24 h); and a TGF-β1 + Y-27632 (a specific ROCK inhibitor) group (pre-treated with 10 µM Y-27632 for 2 h and subsequently treated with 5 ng/ml TGF-β1 for 24 h). The levels of ZO-1 and occludin were detected by real-time PCR, western blot analysis and immunofluorescence. As shown by our results, the mRNA and protein levels of ZO-1 and occludin were decreased in the HK-2 cells following treatment with TGF-β1 in a time-dependent manner; in addition, ZO-1 and occludin levels in the TGF-β1 + Y-27632 group were significantly increased compared with those of the TGF-β1 group (P<0.05), with no significant changes compared with the control group. Our results indicate that the Rho/ROCK signaling pathway mediated by TGF-β1 plays a role in the dissolution of TJs during EMT.