Ras homolog gene family, member A (RhoA) is a small GTPase protein known to regulate multiple cellular processes. In the present study, we used both an alcohol-fed mouse model and an alcohol-treated Caco-2 intestinal epithelial cell monolayer in vitro model to investigate whether RhoA is involved in alcohol-induced intestinal barrier dysfunction as well as the underlying mechanisms. We found that chronic alcohol exposure significantly increased both intestinal RhoA mRNA and protein levels in mice and alcohol treatment also increased RhoA activity in Caco-2 cells. The alcohol-induced elevation in RhoA activity was accompanied by an increase in inducible nitric oxide synthase (iNOS) expression and prevented by N⁶-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL) or small interfering RNA (siRNA) specific for iNOS. Furthermore, alcohol treatment with Caco-2 cells resulted in a significant decrease in the epithelial transepithelial electrical resistance (TEER) value, which was attenuated by knockdown of RhoA. Taken together, our findings suggest that iNOS-mediated activation of RhoA appears to be one of the important mechanisms contributing to the deleterious effects of alcohol on intestinal barrier function.