In this work, we investigated the potential role of the small G protein RhoA in ethanol-induced tight junction (TJ) protein disassembly and increased intestinal epithelial barrier (IEB) permeability. Our study used Caco-2 cells as an in vitro IEB model and RhoA short hairpin RNA (shRNA) interference to establish whether RhoA plays a role in ethanol-induced TJ opening. RhoA shRNA interference partially inhibited epithelial leakage and restored normal transepithelial electrical resistance (TEER) values in the IEB. Moreover, RhoA shRNA interference prevented a shift in occludin distribution from insoluble to soluble fractions. Additionally, RhoA shRNA interference inhibited the ethanol-induced expression of zonula occludens-1 (ZO-1). Finally, RhoA shRNA interference inhibited an ethanol-induced increase in RhoA activity. The contributions of RhoA to an ethanol-induced increase in IEB permeability are associated with TJ disassembly.