Adult hepatic progenitor (oval) cells are facultative stem cells in liver, which participate in a range of human liver diseases, including hepatocellular carcinoma (HCC). However, the molecular pathways regulating the expansion and differentiation of these cells are poorly understood. We show that active Wnt/β-catenin signaling occurs preferentially within the oval cell population, and forced expression of constitutively active β-catenin mutant promotes expansion of the oval cell population in the regenerated liver. More importantly, we identify a subpopulation of less differentiated progenitor-like cells in HCC cell lines and primary HCC tissues, which are defined by expression of the hepatic progenitor marker OV6 and endowed with endogenously active Wnt/β-catenin signaling. These OV6⁺ HCC cells possess a greater ability to form tumor in vivo and show a substantial resistance to standard chemotherapy compared with OV6⁻ tumor cells. The fraction of tumor cells expressing OV6 is enriched after Wnt pathway activation, whereas inhibition of β-catenin signaling leads to a decrease in the proportion of OV6⁺ cells. In addition, the chemoresistance of OV6⁺ HCC progenitor-like cells can be reversed by lentivirus-delivered stable expression of microRNA targeting β-catenin. These results highlight the importance of the Wnt/β-catenin pathway in activation and expansion of oval cells in normal rodent models and human HCCs. OV6⁺ tumor cells may represent the cellular population that confers HCC chemoresistance, and therapies targeted to the Wnt/β-catenin signaling may provide a specific method to disrupt this resistance mechanism to improve overall tumor control with chemotherapy. [Cancer Res 2008;68(11):4287–95]