Enterohaemorrhagic Escherichia coli (EHEC), serotype O157: H7 is a non-invasive, pathogenic bacterium that employs a type III secretion system (T3SS) to inject effector proteins into infected cells. In this study, we demonstrate that EHEC blocks tumour necrosis factor-alpha (TNFa)- induced NF-κB signalling in infected epithelial cells. HEK293T and INT407 epithelial cells were challenged with EHEC prior to stimulation with TNFa. Using complementary techniques, stimulation with TNFa caused activation of NF-κB, as determined by luciferase reporter assay (increase in gene expression), Western blotting (phosphorylation of IκBα), immunofluorescence (p65 nuclear translocation) and immunoassay (CXCL-8 secretion), and each was blocked by EHEC O157: H7 infection. In contrast, subversion of host cell signalling was not observed following exposure to either enteropathogenic E. coli, strain E2348/69 (O127: H6) or the laboratory E. coli strain HB101. Heat-killed EHEC had no effect on NF-κB activation by TNFα. Inhibition was mediated, at least in part, by Shiga toxins and by the O157 plasmid, but not by the T3SS or flagellin, as demonstrated by using isogenic mutant strains. These findings indicate the potential for developing novel therapeutic targets to interrupt the infectious process.