Role of l-glutamine in the protection of intestinal epithelium from acetaldehyde-induced disruption of barrier function was evaluated in Caco-2 cell monolayer. l-Glutamine reduced the acetaldehyde-induced decrease in transepithelilal electrical resistance and increase in permeability to inulin and lipopolysaccharide in a time- and dose-dependent manner; d-glutamine, l-aspargine, l-arginine, l-lysine, or l-alanine produced no significant protection. The glutaminase inhibitor 6-diazo-5-oxo-l-norleucine failed to affect the l-glutamine-mediated protection of barrier function. l-Glutamine reduced the acetaldehyde-induced redistribution of occludin, zonula occludens-1 (ZO-1), E-cadherin, and β-catenin from the intercellular junctions. Acetaldehyde dissociates occludin, ZO-1, E-cadherin, and β-catenin from the actin cytoskeleton, and this effect was reduced by l-glutamine. l-Glutamine induced a rapid increase in the tyrosine phosphorylation of EGF receptor, and the protective effect of l-glutamine was prevented by AG1478, the EGF-receptor tyrosine kinase inhibitor. These results indicate that l-glutamine prevents acetaldehyde-induced disruption of the tight junction and increase in the paracellular permeability in Caco-2 cell monolayer by an EGF receptor-dependent mechanism.