Wnt/␤-catenin signaling contributes to diverse cellular functions, such as Drosophila wing development and colon carcinogenesis. Recently, stabilizing mutations of ␤-catenin, a hallmark of Wnt signaling, were documented in significant numbers of primary hepatocellular carcinomas (HCC). However, whether the ␤-catenin mutation leads to the activation of Wnt/␤-catenin signaling in hepatoma cells has not been established. We found that Wnt/ ␤-catenin signaling could be activated by ectopic expression of Wnt-1 in some hepatoma cells, such as Hep3B and PLC/PRF/5 cells, but not in others, such as Huh7 and Chang cells. Importantly, we noted that the former were derived from hepatitis B virus (HBV)-infected livers, whereas the latter were derived from HBV-negative livers. It was then speculated that HBx, a viral regulatory protein of HBV, is involved in activating Wnt/␤-catenin signaling in hepatoma cells. In agreement with this notion, ectopic expression of HBx along with Wnt-1 activated Wnt/␤-catenin signaling in Huh7 cells by stabilizing cytoplasmic ␤-catenin. Further, we showed that such stabilization of ␤-catenin by HBx was achieved by suppressing glycogen synthase kinase 3 activity via the activation of Src kinase. In conclusion, the data suggest that Wnt-1 is necessary but insufficient to activate Wnt/␤-catenin signaling in hepatoma cells and the enhanced stabilization of ␤-catenin by HBx, in addition to Wnt-1, is essential for the activation of Wnt/␤-catenin signaling in hepatoma cells. (HEPATOLOGY 2004;