Helicobacter pylori induces NF-κB activation, leading to mucosal inflammation via cag pathogenicity island. Although recent studies have implicated several candidate proteins of both H. pylori and host, the molecular mechanism by which H. pylori activates NF-κB remains unclear. The aim of this study was to analyze the mechanism of cag pathogenicity island-mediated NF-κB activation in epithelial cells. The responses of human cell lines and mouse embryonic fibroblasts to infection with wild-type H. pylori or cagE mutant were investigated. The effect of small interfering RNAs (siRNAs) for several NF-κB signaling intermediate molecules was evaluated in H. pylori-induced IκBα phosphorylation and IL-8 production. Protein interactions of exogenously expressed TNFR-associated factor 6 (TRAF6) and MyD88 or receptor-interacting protein 2 and nucleotide-binding oligomerization domain 1 or those of endogenous IκB kinase, TGF-β-activated kinase 1 (TAK1), and TRAF6 were assessed by immunoprecipitation. Cag pathogenicity island-dependent NF-κB activation was observed in human cell lines, but not in mouse fibroblasts. In human epithelial cells, H. pylori-induced IκBα phosphorylation and IL-8 production were severely inhibited by siRNAs directed against TAK1, TRAF6, and MyD88. In contrast, siRNAs for TRAF2, IL-1R-associated kinases 1 and 4, and cell surface receptor proteins did not affect these responses. H. pylori infection greatly enhanced MyD88 and TRAF6 complex formation in a cag-dependent manner, but did not enhance Nod1 and receptor-interacting protein 2 complex formation. H. pylori also induced TAK1 and TRAF6 complexes. These results suggest that the cag pathogenicity island of H. pylori is a cell type-specific NF-κB activator. TAK1, TRAF6, and MyD88 are important signal transducers in H. pylori-infected human epithelial cells.