Enterohaemorrhagic Escherichia coli O157:H7 (EHEC) is a gastrointestinal pathogen that is generally non-invasive for intestinal epithelial cells, yet causes acute intestinal inflammation, diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome. To study signal transduction pathways activated in human intestinal epithelial cells by EHEC, we took advantage of EHEC O157:H7 and isogenic mutants deficient in the major EHEC virulence factors, intimin (eae–) and Shiga toxin (stx–). Infection with wild-type EHEC activated p38 and ERK MAP kinases and the nuclear translocation of the transcription factor NF-κB. Downstream, this was accompanied by increased expression of mRNA and protein for the neutrophil chemoattractant IL-8. Isogenic eae– and stx– mutants also activated p38 and ERK MAP kinases, and NF-κB and stimulated increases in IL-8 protein secretion similar to those of wild-type EHEC. Further, inhibition of either p38, ERK or NF-κB activation abrogated the IL-8 response induced by wild-type EHEC and the mutants. Epithelial cell MAP kinase and NF-κB pathways leading to IL-8 secretion were also activated by isolated EHEC H7 flagellin, which was active when added to either the apical or basolateral surface of polarized human intestinal epithelial cells. We conclude that EHEC interacting with intestinal epithelial cells activates intracellular signalling pathways and an epithelial cell proinflammatory response independent of either Shiga toxin or intimin, two of the major known virulence factors of EHEC. The activation of proinflammatory signals in human colon epithelial cells in response to this non-invasive pathogen appears to depend to a significant extent on H7 flagellin.