After intramembranous proteolysis-mediated loss of the extracellular domain of the epithelial cell adhesion molecule (EpEx) and release of an intracellular domain (EpICD) into the cytoplasm, EpICD sequentially associates with FHL2 to form a nuclear complex with β-catenin and Lef-1. This association induces gene transcription involved in the activation of the oncogenic potential of epithelial cell adhesion molecule (EpCAM). We examined the localization and expression of EpEx, EpICD and β-catenin in surgical specimens of extrahepatic cholangiocarcinoma (ECC) from 79 patients and focused on the relationship between nuclear expression of EpICD and β-catenin. We also examined the role of EpICD by transfecting the EpICD cDNA in cholangiocarcinoma (CC) cell lines. There was a significant correlation between the nuclear expression of EpICD and β-catenin in ECC tissues. Frequent nuclear co-localization of EpICD and β-catenin was observed in cancer cells forming the invasive front. Nuclear expression of EpICD also significantly correlated with histologic grade of tumor. Overexpression of EpICD in the CC cells significantly increased the cell growth and proliferation. The overexpression of EpICD in the CC cells also increased the expression levels of the active form of β-catenin and EpCAM target genes, such as c-myc and cyclin D1. Furthermore, the overexpression of EpICD significantly enhanced the migration and invasiveness of CC cells. Conversely, the inhibition of EpCAM in EpCAM-overexpressing cells by siRNA significantly decreased cell proliferation, migration and invasion. These results indicate that the spatial localization of EpICD and its mutual interaction with β-catenin may be important in ECC progression and invasion.