<p>Pyroptosis contributes to activation of the innate immunity system and defense against infection by pathogens. Endotoxemia is the host inflammatory storm occurring in response to severe and life-threatening infections caused by endotoxin from gram-negative bacilli. However, whether pyroptosis is involved in intestinal epithelial cell (IEC) or intestinal stem cell (ISC) injury induced by endotoxemia remains unclear. Mice with NF-κB p65 deletion in IECs (p65^{IEC − KO}) were used to investigate the role of NF-κB-mediated pyroptosis in endotoxemia-induced intestinal injury. Morphology, pyroptosis, permeability, inflammation, endoplasmic reticulum stress in the intestine and survival were evaluated in WT and p65^{IEC − KO} mice. Pyroptosis was found in intestinal epithelial cells of mice treated with lipopolysaccharide (LPS), but was significantly reduced in p65^{IEC − KO} mice. Mice with endotoxemia exhibited morphological alterations in intestinal tissue, with a shortened villus length and crypt depth, increased intestinal permeability, increased inflammatory factors, and reduced survival rate, all of which were markedly improved in p65^{IEC − KO} mice. Importantly, ER stress was found to be downregulated in IECs of p65^{IEC − KO} mice with endotoxemia. Furthermore, the ER stress activator tunicamycin markedly enhanced IEC pyroptosis and aggravated intestinal injury in p65^{IEC − KO} mice with endotoxemia. NF-κB p65–mediated pyroptosis participates in IEC injury in response to endotoxemia via regulation of ER stress. It may provide a potential therapeutic target for protecting against endotoxemia-induced intestinal injury.</p>