UNC119 (uncoordinated 119 or retinal protein 4), specifically expressed in the photoreceptors in the retina, has recently been found to be upregulated in hepatocellular carcinoma (HCC) tissues, predicting a poor prognosis. However, the biological role of UNC119 in cancer treatment is still poorly understood. Gambogic acid (GA), a major component of gambogic resin, has been shown to possess anticancer activity against multiple human cancer cell lines. In the present study, we discovered that GA was more effective in inhibiting cell proliferation in HCC cells with a higher level of UNC119. In addition, GA inhibited UNC119 expression and induced Hep3B cells G0/G1 arrest. Cell-cycle-related proteins, such as cyclin A, E, D1, and p-cyclin-dependent kinase 2, 4, 6 were downregulated in GA-treated cells. Glycogen synthase kinase 3β (Gsk3β)/β-catenin signaling, the downstream of UNC119, was also found to be suppressed after GA treatment. UNC119 knockdown or over expression experiment further proved that UNC119 mediated the effect of GA on the HCC cell cycle and Gsk3β/β-catenin signaling. In BALB/c mice bearing xenotransplanted tumors, the growth of the Hep3B tumor was inhibited by GA treatment. Immunohistochemistry results of tumor tissues suggested that GA might also exert its anticancer effect by inhibiting UNC119 and regulating cell cycle in vivo. Thus, GA could be a potential therapeutic agent in the treatment of human HCC.