Helicobacter pylori infection has been proposed to be associated with various diseases of the hepatobiliary tract, including cancer of the bile duct epithelial cells (cholangiocarcinoma, CCA). The ability of H. pylori bacteria to cause pathogenic effects in these cells has, however, yet to be investigated. Given that the cag pathogenicity island (cagPAI) is required for H. pylori pathogenesis in gastric epithelial cells, we investigated wild-type and cag mutant strains for their ability to adhere, be internalized and induce pro-inflammatory responses in two bile duct epithelial cell lines derived from cases of CCA. The findings from these experiments were compared to results obtained with the well-characterized AGS gastric cancer cell line. We showed that the cagPAI encodes factors involved in H. pylori internalization in CCA cells, but not for adhesion to these cells. Consistent with previous studies in hepatocytes, actin polymerization and α5β1 integrin may be involved in H. pylori internalization in CCA cells. As for AGS cells, we observed significantly reduced levels of NF-κB activation and IL-8 production in CCA cells stimulated with either cagA, cagL or cagPAI bacteria, when compared with wild-type bacteria. Importantly, these IL-8 responses could be inhibited via either pre-treatment of cells with antibodies to α5β1 integrins, or via siRNA-mediated knockdown of the innate immune signaling molecules, nucleotide oligomerization domain 1 (NOD1) and myeloid differentiation response gene 88 (MyD88). Taken together, the data demonstrate that the cagPAI is critical for H. pylori pathogenesis in bile duct cells, thus providing a potential causal link for H. pylori in biliary tract disease.