UNLABELLED Cholangiocarcinoma (CCA) is a lethal hepatobiliary neoplasm originating from the biliary apparatus. In humans, CCA risk factors include hepatobiliary inflammation and fibrosis. The recently identified interleukin (IL)-1 family member, IL-33, has been shown to be a biliary mitogen which also promotes liver inflammation and fibrosis. Our aim was to generate a mouse model of CCA mimicking the human disease. Ectopic oncogene expression in the biliary tract was accomplished by the Sleeping Beauty transposon transfection system with transduction of constitutively active AKT (myr-AKT) and Yes-associated protein. Intrabiliary instillation of the transposon-transposase complex was coupled with lobar bile duct ligation in C57BL/6 mice, followed by administration of IL-33 for 3 consecutive days. Tumors developed in 72% of the male mice receiving both oncogenes plus IL-33 by 10 weeks but in only 20% of the male mice transduced with the oncogenes alone. Tumors expressed SOX9 and pancytokeratin (features of CCA) but were negative for HepPar1 (a marker of hepatocellular carcinoma). Substantive overlap with human CCA specimens was revealed by RNA profiling. Not only did IL-33 induce IL-6 expression by human cholangiocytes but it likely facilitated tumor development in vivo by an IL-6-sensitive process as tumor development was significantly attenuated in Il-6(-/-) male animals. Furthermore, tumor formation occurred at a similar rate when IL-6 was substituted for IL-33 in this model. CONCLUSION The transposase-mediated transduction of constitutively active AKT and Yes-associated protein in the biliary epithelium coupled with lobar obstruction and IL-33 administration results in the development of CCA with morphological and biochemical features of the human disease; this model highlights the role of inflammatory cytokines in CCA oncogenesis.