The objective of this study was to investigate the role of the RhoA/Rock signaling pathway in the epithelial-mesenchymal transition (EMT) of rat peritoneal mesothelial cells (RPMCs). Primary SD rat peritoneal mesothelial cells were cultured in vitro. RPMCs were randomly assigned to four groups: group A (control), group B (TGF-β1, 10 μg/L), group C (10 μg/L TGF-β1 + 10 μmol/L Y-27632, an inhibitor of Rock that was pre-applied for 2 h before TGF-β1 stimulation), and group D (Y-27632 alone, 10 μmol/L). Our results were as follows: (1) TGF-β1 stimulation elicited a robust increase in RhoA activity in a time-dependent manner; the increase was 2.57 ± 0.52 times larger than the activity observed for the control group (P < 0.05) after 10 min of stimulation. RhoA activity peaked at 1 h and was 4.35 ± 0.41 times the value observed for the control group (P < 0.05). (2) TGF-β1 up-regulated mRNA and/or protein expression of α-SMA, vimentin, and collagen and down-regulated mRNA and protein expression of E-cadherin in RPMCs. (3) The Rock inhibitor Y-27632 effectively reduced TGF-β1-induced expression of α-SMA, collagen, and vimentin; the mRNA levels of α-SMA and collagen decreased by 53.8% and 55.7%, respectively, and the protein levels of α-SMA, vimentin, and collagen decreased by 42.6%, 60.1%, and 58.1%, respectively, as compared to TGF-β1-stimulated groups (P < 0.05). However, the Rock inhibitor Y-27632 had no effect on the level of E-cadherin. In conclusion, the RhoA/Rock signaling pathway may mediate EMT induced by TGF-β1 in rat peritoneal mesothelial cells. The RhoA/Rock pathway may be a potential therapeutic target for the treatment of peritoneal fibrosis.