Introduction: Impaired intestinal epithelial barrier function is a hallmark of a variety of pathological conditions such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). IBD patients with IBS-like symptoms show higher interleukin-13 (IL-13) serum levels and poor psychological well-being. Supplementary glutamine reduced the daily bowel movement frequency, improved the stool form, and normalized intestinal hyperpermeability. This study was aimed at assessing the effects of IL-13 and supplementary glutamine on human intestinal epithelial function in vitro. Methods: Caco-2 cells were grown on TranswellTM inserts. IL-13 was added to the basolateral compartment, and transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) labeled-dextran permeability measured. Effects of glutamine or the phosphatidylinositol-3-kinase inhibitor LY294002 were assessed. Involvement of tight junction proteins was assessed using Western blotting and immunofluorescence staining. Results: IL-13 significantly decreased TEER and increased FITC labeled-dextran epithelial permeability. IL-13 stimulation decreased the claudin-1 expression and increased the claudin-2 expression. Glutamine alleviated IL-13-induced decrease of TEER and increase of FITC labeled-dextran permeability. Further, the phosphatidylinositol-3-kinase inhibitor showed this alleviating effect while the signal transducer and activator of transcription 6 inhibitor did not. Conclusions: IL-13 induced barrier integrity impairment by decreasing claudin-1 and increasing claudin-2. Glutamine alleviated IL-13-induced barrier dysfunction by increasing claudin-1 expression, via disruption of the phosphatidylinositol-3-kinase/Akt signaling pathway.