Hepatocellular carcinoma (HCC) is one of the most aggressive malignant tumors, of which treatment options are limited especially in advanced stage. Bufalin, the major digoxin-like component of the traditional Chinese medicine Chansu, exhibits significant antitumor activities in hepatoma cells, but the potential mechanism is obscure. Cell cycle-related kinase (CCRK) is recently identified to be a crucial oncogenic master regulator to drive hepatocarcinogenesis. Here we investigated the molecular function of bufalin on CCRK-regulated signaling pathway, and expounded the underlying mechanism in HCC suppression. In vitro with PLC5 HCC cells and human immortal LO2 cells, proliferation, malignant transformation and cell cycle progression assays were performed to evaluate the antitumor effect of bufalin. In vivo with xenograft and orthotopic mice models, tumor growths with weight and volume change were assessed with or without bufalin treatment. Western blot, RT-qPCR, immunofluorescence and immunohistochemistry were conducted to examine the expression level of CCRK and β-catenin/TCF signaling cascade. We revealed that bufalin suppresses PLC5 HCC cell proliferation, transformation and cell cycle progression rather than LO2 cells, which is correlated with CCRK-mediated β-catenin/TCF signaling. It was also confirmed in mice model. Thus, bufalin is a potential anti-HCC therapeutic candidate through the inhibition of CCRK-driven β-catenin/TCF oncogenic signaling pathway.