Background & aims: HNF4-alpha expression is associated with tumor suppression in hepatocellular carcinoma (HCC), but the molecular mechanisms involved in this signaling pathway remain elusive. Methods: HNF4-alpha expression was investigated by Western blot and immunohistochemistry (IHC) analyses in HCC cell lines and tissues, respectively. Colony forming ability and tumorigenicity were examined in vitro and in a mouse model system through studies of gain or loss of function. Molecular mechanisms of this signaling pathway were explored. Results: HNF4-alpha expression inhibited the tumorigenicity of SH-J1 cells and its knockdown enhanced tumorigenicity of Alexander (AXL) cells, in vitro and in a xenotransplanted mouse model. Transcription factor array data revealed that FoxH1 expression increased in the nucleus of AXL cells with HNF4α knockdown. The transcription factor, FoxH1, was shown to be inversely regulated by HNF4-alpha expression in HCC cells. AXL cells stably expressing FoxH1 grew fast in vitro and were more tumorigenic in the animal model system. SK-HEP-1 cells with FoxH1 knockdown grew slowly and were less tumorigenic than control cells. FoxH1 was associated with β-catenin activation in vitro. Therefore, HNF4-alpha suppressed β-catenin activation through the inhibition of FoxH1 expression as a tumor suppressor. Conclusions: HNF4-α suppresses hepato-oncogenesis through inhibition of FoxH1-mediated β-catenin activation. These downstream signaling molecules may be potential therapeutic targets in HCC.