The lipopeptides produced by Bacillus subtilis have anti-cancer potential. We had previously identified a secondary metabolite of B. subtilis strain Z15 (BS-Z15), which has an operon that regulates lipopeptide synthesis, and also demonstrated that the fermentation products of this strain exerted antioxidant and pro-immune effects. The purpose of this study was to investigate in vitro and in vivo the anticancer effects of BS-Z15 secondary metabolites (BS-Z15 SMs) on hepatocellular carcinoma (HCC) cells. BS-Z15 SMs significantly inhibited H22 cell-derived murine xenograft tumor growth without any systemic toxicity. In addition, BS-Z15 SMs decreased the viability of H22 cells and BEL-7404 cells in vitro with respective IC