Glutamine is a key amino acid in the central nervous system (CNS), playing an important role in the glutamate/GABA-Glutamine cycle (GGC). In the GGC,Glutamine is transferred from astrocytes to neurons, where it will replenish the inhibitory and excitatory neurotransmitter pools. D-Glutamine has been used to study its role in conferring protection against acetaldehyde-induced disruption of barrier function in Caco-2 cell monolayer. Role of L-Glutamine in the protection of intestinal epithelium from acetaldehyde-induced disruption of barrier function is 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. D-Glutamine also fails to influence the acetaldehyde-induced decrease in TER and increase in inulin flux. D-Glutamine or glutaminase inhibitor by themselves did not influence TER or inulin flux in control or acetaldehyde-treated cell monolayers. Lack of effect of D-Glutamine in protection from acetaldehyde indicates that the L-Glutamine-mediated protection is stereospecific.