Mice which lack the p50 subunit of NF-κB and are heterozygous for the p65 subunit (3X mice), are exquisitely sensitive to LPS-induced shock. Here, we demonstrate that prior to becoming moribund, 3X mice challenged with LPS develop a profound enteropathy. The enteropathy is characterized by defects in intestinal barrier function, increased epithelial apoptosis, and deregulated intestinal cytokine gene expression. The defect that sensitizes 3X mice to LPS-induced enteropathy is located within the innate immune compartment, as LPS induced similar findings in 3X mice lacking lymphocytes (3X/RAG). TNF-α depletion ameliorated the ability of LPS to induce pathology and TNF-α was able to independently induce similar findings, suggesting that TNF-α plays a critical role in the development of LPS-induced pathology in these mice. These data highlight that NF-κB subunits have essential functions in regulating intestinal homeostasis during acute inflammation.