Recent studies suggest that the ability to form and grow tumors specifically resides in a small cell population called cancer stem cells (CSCs). These studies were conducted mainly on various human cancers; however, isolation and characterization of stem cells from cholangiocarcinoma have not been attempted. The molecular markers CD24, CD44, CD34, and epithelial cell adhesion molecule (EpCAM) are widely used, individually or in combination, to characterize some types of CSCs. In this study, we used these markers to identify a subpopulation of cells in extrahepatic cholangiocarcinoma (ECC) with cancer stem/progenitor cell-like properties. We found that CD24+CD44+EpCAMhigh cells (0.39–2.27%) were present in human ECC tissues. The expression of a CD24+CD44+EpCAMhigh subpopulation was consistent with primary cancers and could be duplicated during serial in vivo passaging in NOD/SCID mice. CD24+CD44+EpCAMhigh cells isolated from 3 cholangiocarcinoma xenografts showed high tumorigenic potential compared with CD24−CD44−EpCAMlow/− cells. These tumorigenic ECC cells exhibited the stem cell properties of self-renewal and ability to produce heterogeneous progeny. We report the identification of a CSC population in ECC characterized by CD24, CD44 and EpCAM phenotypes. Our findings could provide new insight into the tumorigenesis of cholangiocarcinoma and offer a potential target for anti-cancer therapy.