Deregulation of intestinal immune responses seems to have a principal function in the pathogenesis of inflammatory bowel disease. The gut epithelium is critically involved in the maintenance of intestinal immune homeostasis-acting as a physical barrier separating luminal bacteria and immune cells, and also expressing antimicrobial peptides. However, the molecular mechanisms that control this function of gut epithelial cells are poorly understood. Here we show that the transcription factor NF-kappaB, a master regulator of pro-inflammatory responses, functions in gut epithelial cells to control epithelial integrity and the interaction between the mucosal immune system and gut microflora. Intestinal epithelial-cell-specific inhibition of NF-kappaB through conditional ablation of NEMO (also called IkappaB kinase-gamma (IKKgamma)) or both IKK1 (IKKalpha) and IKK2 (IKKbeta)-IKK subunits essential for NF-kappaB activation-spontaneously caused severe chronic intestinal inflammation in mice. NF-kappaB deficiency led to apoptosis of colonic epithelial cells, impaired expression of antimicrobial peptides and translocation of bacteria into the mucosa. Concurrently, this epithelial defect triggered a chronic inflammatory response in the colon, initially dominated by innate immune cells but later also involving T lymphocytes. Deficiency of the gene encoding the adaptor protein MyD88 prevented the development of intestinal inflammation, demonstrating that Toll-like receptor activation by intestinal bacteria is essential for disease pathogenesis in this mouse model. Furthermore, NEMO deficiency sensitized epithelial cells to tumour-necrosis factor (TNF)-induced apoptosis, whereas TNF receptor-1 inactivation inhibited intestinal inflammation, demonstrating that TNF receptor-1 signalling is crucial for disease induction. These findings demonstrate that a primary NF-kappaB signalling defect in intestinal epithelial cells disrupts immune homeostasis in the gastrointestinal tract, causing an inflammatory-bowel-disease-like phenotype. Our results identify NF-kappaB signalling in the gut epithelium as a critical regulator of epithelial integrity and intestinal immune homeostasis, and have important implications for understanding the mechanisms controlling the pathogenesis of human inflammatory bowel disease.