Background and Aims: A defective intestinal epithelial tight junction (TJ) barrier has been proposed as an important pathogenic factor contributing to the intestinal inflammation of Crohn's disease. Glucocorticoids are first-line therapeutic agents for the treatment of Crohn's disease. Glucocorticoid treatment has been shown to induce re-tightening of the TJ barrier defect in CD patients. This study's aim was to elucidate the mechanism of glucocorticoid modulation of the intestinal epithelial TJ barrier. Methods: Caco-2 intestinal epithelial cells were used as an in-vitro model to examine the effects of glucocorticoids on basal TJ barrier function and on tumor necrosis factor- (TNF-) induced disruption of the tight junction barrier. Results: Glucocorticoids did not have a significant effect on baseline Caco-2 TJ barrier function but prevented the TNF- induced increase in TJ permeability. The glucocorticoid protective effect against the TNF- induced increase permeability required activation of the glucocorticoid receptor (GR) complex. The activation of the GR complex resulted in binding to the glucocorticoid response element (GRE) site on DNA. Glucocorticoids inhibited the TNF- induced increase in myosin light chain kinase (MLCK) protein expression, mediating the TNF- increase in intestinal TJ permeability. Glucocorticoid inhibition of the TNF- induced increase in MLCK expression was due to the binding of the GR complex to a GRE binding site on the MLCK promoter suppressing TNF- induced activation. Conclusions: Glucocorticoids inhibit the TNF- induced increase in Caco-2 TJ permeability. The prednisolone protective action was mediated by binding of activated GR complex to the GRE site on the MLCK promoter.