Acute injury to the intestinal mucosa is a major dose-limiting complication of abdominal radiation therapy. We studied the role of the transcription factor NF-kappaB in protection against radiation-induced apoptosis in the intestinal epithelium in vivo. We use mice in which NF-kappaB signaling through IkappaB-kinase (IKK)-beta is selectively ablated in intestinal epithelial cells to show that failure to activate epithelial cell NF-kappaB in vivo results in a significant increase in radiation-induced epithelial cell apoptosis. Furthermore, bacterial lipopolysaccharide, which is normally a radioprotective agent, is radiosensitizing in IKKbeta-deficient intestinal epithelial cells. Increased apoptosis in IKKbeta-deficient intestinal epithelial cells was accompanied by increased expression and activation of the tumor suppressor p53 and decreased expression of antiapoptotic Bcl-2 family proteins. These results demonstrate the physiological importance of the NF-kappaB system in protection against radiation-induced death in the intestinal epithelium in vivo and identify IKKbeta as a key molecular target for radioprotection in the intestine. Selective preactivation of NF-kappaB through IKKbeta in intestinal epithelial cells could provide a therapeutic modality that allows higher doses of radiation to be tolerated during cancer radiotherapy.