The purpose of this study was to investigate the clinicopathological and biological significance of human carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) gene expression in human intrahepatic cholangiocarcinoma. CEACAM6 is reported to be involved in human malignancies. However, in cholangiocarcinoma expression of CEACAM6 and its clinicopathological significance have not been investigated. CEACAM6 expression status was determined and analysed with respect to various clinicopathological parameters in 23 intrahepatic cholangiocarcinomas. Additionally, we investigated effects of CEACAM6 gene in the cholangiocarcinoma cell lines. CEACAM6 gene expression in cancer tissues was higher than in noncancerous tissues in 16 of the 23 cases; however, it was not statistically significant. The tumours with elevated CEACAM6 expression showed a tendency to be associated with lymphatic invasion and stage of the disease. Interestingly, patients with high CEACAM6 expression showed a significantly poorer disease-free survival rate than those with low CEACAM6 expression. We demonstrated that CEACAM6-transfected cells were more proliferative, more invasive and more chemoresistant to gemcitabine compared to mock-transfected cells. Furthermore, CEACAM6 gene silencing by CEACAM6-specific siRNA resulted in higher chemosensitivity to gemcitabine. CEACAM6 is a potential prognostic indicator and potential chemoresistant marker to gemcitabine for patients with intrahepatic cholangiocarcinoma.