Polycomb-group proteins Bmi1 is regarded as a "stemness" gene involved in the maintenance of stem cells, malignant transformation, and biologic aggressiveness of several human carcinomas. We examined the significance of the Bmi1 expression in intrahepatic cholangiocarcinoma. The expression of Bmi1 was examined in intrahepatic cholangiocarcinoma (n = 30; 9 bile ductular carcinoma, 8 intrahepatic cholangiocarcinoma of peripheral type, and 13 of hilar type) by using immunohistochemistry and real-time polymerase chain reaction. The expression level of Bmi1 was assessed in 7 cholangiocarcinoma cell lines. The effect of Bmi1 knockdown was examined in cultured cholangiocarcinoma cells (HuCCT1 and TFK-1) using small interfering RNA. Bmi1 was consistently expressed in nonneoplastic biliary epithelial cells and in all intrahepatic cholangiocarcinoma, irrespective of the location and histological degree of differentiation. The level of mRNA expression was significantly higher in 13 (81.3%) of 16 intrahepatic cholangiocarcinoma compared with the corresponding nonneoplastic tissues. All 7 cultured cholangiocarcinoma cells overexpressed Bmi1 to various degrees. The knockdown of Bmi1 resulted in decreased colony formation, decreased cell proliferation activities, and increased cellular senescence. The overexpression of polycomb-group protein Bmi1 is essential for colony formation and cell proliferation, probably by the repression of cellular senescence in intrahepatic cholangiocarcinoma.