The transcription factor NF-kappaB in human intestinal epithelial cells plays a central role in regulating genes that govern the onset of mucosal inflammatory responses following intestinal microbial infection. Nod1 is a cytosolic pattern recognition receptor in mammalian cells that senses components of microbial peptidoglycans and signals the activation of NF-kappaB. The aim of these studies was to assess the functional importance of Nod1 in activating NF-kappaB and NF-kappaB proinflammatory target genes in human intestinal epithelium. Human colon epithelial cells that constitutively express Nod1 were used as a model intestinal epithelium. These cells do not signal through Toll-like receptor 4 (TLR4) or respond to bacterial lipopolysaccharide, but they express functional TLR5 and interleukin 1 (IL-1) receptors that signal the activation of NF-kappaB in response to bacterial flagellin or IL-1 stimulation. Stable expression of dominant negative (DN) Nod1 in colon epithelial cells prevented IkappaB kinase and NF-kappaB activation in response to infection with enteroinvasive Escherichia coli. In contrast, DN Nod1 did not eliminate IL-1 or flagellin-stimulated NF-kappaB activation. Inhibition of NF-kappaB was accompanied by inhibition of NF-kappaB target genes that provide signals for the mucosal influx of neutrophils during intestinal infection. We conclude that signaling through Nod1 is required for activating NF-kappaB in human intestinal epithelial cells infected with gram-negative enteric bacteria that can bypass TLR activation. Signaling through Nod1 provides the intestinal epithelium with a backup mechanism for rapidly activating innate immunity during infection with a group of highly invasive pathogenic gram-negative bacteria.