Chaetocin is a fungal metabolite that possesses a potent antiproliferative activity in solid tumors by inducing cell death. Although recent studies have extended the role of chaetocin in tumors, the underlying molecular mechanisms such as the downstream cascade that induces cell death has not clearly been elucidated. In this study, we show that chaetocin is able to induce both apoptosis and autophagy in several hepatoma cell lines including HepG2, Hep3B and Huh7 cell lines. Moreover, we found that the inhibition of caspase-3/7 activity by z-VAD-fmk treatment was able to block chaetocin-mediated cell death, whereas blocking autophagy by Bafilomycin A1 or the knockdown of autophagy protein 5 enhanced cell death mediated by chaetocin. These findings suggest that chaetocin has a potent anticancer effect against hepatoma. Inhibition of autophagy may potentiate anticancer effects of chaetocin thus providing evidence that combined treatment with chaetocin and autophagy inhibitors will be an effective strategy for treating cancer.