Vitamin K2 (VK2) exerts cell growth inhibitory effects in various human cancer cells such as SMMC-7721 hepatocellular carcinoma (HCC) cells. BCL-2 is an antiapoptotic protein that is frequently overexpressed in numerous tumors. Modulation of multiple antiapoptotic signaling pathways involving BCL-2, which are related to growth factor-stimulated signal transduction in cell survival, is essential for enhancement of the cytotoxic effect of anticancer drugs. In this study, we tested a new strategy of gene therapy by combining BCL-2 siRNA with VK2. In SMMC-7721 HCC cells, the combined treatment significantly enhanced cytotoxicity compared with treatment with either VK2 or siBCL-2 alone. We found that combined treatment induced a significantly different level of G2 stage inhibition. Furthermore, the p53 protein was overexpressed 24 h subsequent to combination treatment, and p21 was clearly increased at 36 h as a consequence of the increased p53 activity. In conclusion, these data suggest that the antitumor effect of VK2 may be improved by silencing BCL-2 expression in SMMC-7721 HCC cells and provides support for the combined use of VK2 and siBCL-2 as a promising approach in cancer gene therapy.