The use of vincristine, a known antitumor agent, in hepatoma therapy is limited particularly because of its toxic effect. Meanwhile, berberine has drawn increasing attention to its antineoplastic effect in recent years. In view of the advantages of combinational drug treatment reported in anti-cancer chemotherapy, we evaluated the effects of co-treatment of vincristine and berberine on hepatic carcinoma cell lines in this study. We find that combinational usage of these two drugs can significantly induce cell growth inhibition and apoptosis even under a concentration of vincristine barely showing cytotoxicity in the same cells when used alone. The underlying mechanism about this combinational effect was addressed in this study by monitoring the signals related to mitochondrial function, apoptotic pathway and endoplasmic reticulum stress. Our results suggest a new value of berberine as a potential adjuvant agent in cancer chemotherapy and provide a hopeful approach for developing hepatoma therapy by utilizing the combinational effect of vincristine and berberine. J. Cell. Biochem. 115: 721–730, 2014. © 2013 Wiley Periodicals, Inc.