OBJECTIVE Pancreaticobiliary maljunction (PBM) is a high risk factor of biliary tract cancer. The chemopreventive effects of Vitamin K2 (menaquinone-4: MK4) in a hamster PBM model were investigated. METHODS The extrahepatic bile duct at the distal end of the common duct was ligated and cholecystoduodenostomy was performed (Group I). The same surgery was performed and from 4 weeks after surgery, 10 mg/kg of N-nitrosobis (2-oxopropyl) amine was subcutaneously injected once a week with a one-week interval (Group II). In addition of Group II, MK4 was orally administered once a day, five times with every week (Group III). The hamsters were sacrificed 20 weeks after surgery and histopathological findings of gallbladder were investigated. RESULTS Group I showed predominantly proper epithelium without cancer. In Group II, atypical epithelium (AE) was observed in 75% of animals and early cancer was observed in 25%. Group III showed less AE and no cancer. The PCNA labeling index in Group III was statistically significantly lower than in Group II. In addition, no statistically significant differences were noted among the groups in terms of the apoptosis labeling index. CONCLUSIONS MK4 suppressed biliary carcinogenesis by the induction of cell cycle arrest in a hamster biliary carcinogenetic model.