Glutamine (Gln) is important for intestinal barrier function and regulation of tight junction (TJ) proteins, but the intracellular mechanisms of action remain undefined. The purpose of this study was to test the hypothesis that Gln regulates intercellular junction integrity and TJ proteins through the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in Caco-2 cells. Deprivation of exogenous and endogenous glutamine decreased transepithelial electrical resistance (TER) (P < 0.01) and increased permeability (P < 0.01). Both wortmannin and LY294002, PI3K inhibitors, prevented the TER decrease and the permeability increase induced by Gln deprivation (P < 0.001). Gln deprivation also caused decreased TJ protein claudin-1 (P < 0.001). Both wortmannin and LY294002 treatment prevented this effect (P < 0.001). Deprivation of Gln increased phosphor-Akt protein. Gln supplementation reversed this effect. Decreased TER and increased permeability associated with Gln deprivation were not observed in small interfering RNA for p85 transfected Caco-2 cells. In conclusion, Gln regulates intercellular junction integrity and TJ proteins through the PI3-Kinase/Akt pathway.