Intrahepatic cholangiocarcinoma arising in chronic advanced liver disease sometimes contains a component of cholangiolocellular carcinoma. Bile duct adenoma, a benign tumor/tumorous lesion and ductular reaction, is also composed of bile ductular cells, and the differential diagnosis is sometimes difficult. We have previously reported that cholangiolocellular carcinoma showed overexpression of a polycomb group protein EZH2, which participates in bypass/escape from cellular senescence during carcinogenesis. In contrast, the ductular reaction showed high expression of senescence-associated p16(INK4a). In this study, we examined whether immunostaining for EZH2 and p16(INK4a) is useful for differential diagnosis among cholangiolocellular carcinoma, bile duct adenoma, and ductular reactions. Subjects included 33 patients with intrahepatic cholangiocarcinoma and combined hepatocellular-cholangiocarcinoma with components of cholangiolocellular carcinoma and 16 patients with bile duct adenoma. The expressions of EZH2 and p16(INK4a) were examined immunohistochemically. The expression of EZH2 was seen in all cases of cholangiolocellular carcinomas, but it was not observed in bile duct adenomas and ductular reactions, which were seen around carcinomas in 80% of cases. The extensive expression of p16(INK4a) was seen only in 4 cases of cholangiolocellular carcinomas (12%). In contrast, the expression of p16(INK4a) was seen in 13 cases (81%) of bile duct adenomas and in all cases of ductular reactions. The borderline between the component of cholangiolocellular carcinoma and the surrounding ductular reaction was clearly highlighted by the reverse expression pattern of EZH2 and p16(INK4a) in 69% of cases. In conclusion, immunostaining for EZH2 and p16(INK4a) may be useful for differential diagnosis among cholangiolocellular carcinomas, bile duct adenomas, and ductular reactions.