The present study aimed to investigate the effect of Y-27632, an inhibitor of the Rho-associated protein kinase (ROCK), which belongs to a family of downstream effectors of activated RhoA, on the ethanol-induced increase in permeability of the intestinal epithelial barrier (IEB). The in vitro model of IEB was established usiung Caco-2 cells, and the cells were pretreated with Y-27632 prior to treatment with ethanol for 60 min. Transepithelial resistance (TEER) and paracellular marker Lucifer yellow flux measurements were performed to assess the IEB permeability. The localization and expression of tight junction (TJ)-associated proteins were detected by immunofluorescence and western blot analysis, respectively. Y-27632 partially inhibited epithelial leakage and restored normal TEER values in the IEB. Immunofluorescence and western blot analysis results indicated that ethanol induces a shift from the insoluble to the soluble fractions of claudin-1, and that the ethanol-induced decreased expression of the zonula occludens-1 (ZO-1) protein is restored by Y-27632. In conclusion, our results suggest that ROCK may play a key role in the ethanol-induced increase of IEB permeability.