The association between the temporal activation of Wnt/β-catenin pathway and the spontaneous hepatocellular carcinoma (HCC) development in Farnesoid X receptor (FXR) knockout mice is not well understood. We found that Huh7 cells depleted with FXR by RNAi showed enhanced cell growth, migration and invasion in vitro and accelerated tumor xenografts formation in nude mice. And these phenotypes were attenuated by simultaneous knockdown of β-catenin with RNAi. Furthermore, we identified that FXR could bind with β-Catenin through AF1 domain, and disrupt the assembly of the core β-Catenin/TCF4 complex. Activation of FXR attenuated the DNA-binding activity of β-Catenin/TCF4, and subsequently, its targeting gene-cyclin D1 expression. Importantly, FXR expression was markedly reduced in human HCC, an event which correlated with aberrant activation of β-Catenin. These data identified FXR as a negative regulator of HCC development through direct suppression of Wnt/β-catenin pathway.