In inflammatory bowel disease (IBD), aberrant activation of innate and adaptive immune responses enhances mucosal permeability through mechanisms not completely understood. To examine the role of epithelial nuclear factor (NF-κB) in IBD-induced enhanced permeability, epithelial-specific IκBα mutant (NF-κB super repressor) transgenic (TG) mice were generated. NF-kB activation was inhibited in TG mice, relative to wild-type mice, following T cell-mediated immune cell activation using an anti-CD3 monoclonal antibody. Furthermore, epithelial NF-κB super repressor protein inhibited diarrhea and blocked changes in transepithelial resistance and transmucosal flux of alexa350 (0.35 kDa) and dextran3000 (3 kDa). In vivo perfusion loop studies in TG mice revealed reversed net water secretion and reduced lumenal flux of different molecular probes (bovine serum albumin, alexa350, and dextran3000). Cell-imaging and immunoblotting of low-density, detergent-insoluble membrane fractions confirmed that tight junction proteins (occludin, claudin-1 and zona occludens-1) are internalized through an NF-κB-dependent pathway. Taken together, these data suggest that IBD-associated diarrhea results from NF-κB-mediated tight junction protein internalization and increased paracellular permeability. Thus, reduction of epithelial NF-κB activation in IBD may repair defects in epithelial barrier function, reduce diarrhea, and limit protein (eg, serum albumin) losses. Epithelial NF-κB activation induced by mucosal T cells, therefore, actively plays a role in opening paracellular spaces to promote transmucosal fluid effux into the intestinal lumen. Copyright © American Society for Investigative Pathology.