Hwang-Heuk-San (HHS) is a polyherbal formulation that has been used in traditional Korean medicine for hundreds of years to treat gastrointestinal malignancy. However, to date, the mechanisms responsible for the anticancer effects remain unclear. In the present study, we investigated the anticancer effects of HHS using HCT116 human colorectal cancer (CRC) cells. Our results showed that HHS treatment significantly reduced cell survival and increased apoptotic cell death in a concentration-dependent manner. The treatment of HCT116 cells with HHS also significantly elevated the accumulation of reactive oxygen species (ROS), which was followed by the attenuation of the mitochondrial membrane potential through the upregulation of Bax and the downregulation of Bcl-2, which was accompanied by the release of cytochrome c to the cytosol. In addition, HHS treatment caused the truncation of Bid and activated the caspases (caspase-8, -9 and -3), which was associated with the induction of the Fas ligand, the death receptors (DRs), DR4 and DR5, downregulation of the inhibitors of protein expression in the apoptosis protein family, and the degradation of poly(ADP-ribose)-polymerase. However, a pan-caspase inhibitor reversed the HHS-induced apoptosis and growth suppression, indicating that HHS induces apoptosis though a caspase-dependent intrinsic and extrinsic apoptotic pathway in HCT116 cells. Moreover, HHS treatment inhibited the activation of phosphatidylinositol-3-kinase (PI3K)/Akt signaling, and a pharmacological inhibitor of PI3K significantly potentiated the apoptotic effects of HHS when employed in combination in HCT116 cells. Furthermore, the blocking of ROS generation by antioxidant N-acetyl cysteine attenuated the HHS-induced release of cytochrome c, caspase activation and PI3K/Akt inactivation, thereby preventing HHS-induced apoptosis and reduction in cell viability. These findings suggest that HHS-induced ROS generation is required for caspase-dependent apoptotic cell death involving inhibition of the PI3K/Akt signaling pathway in HCT116 cells. Overall, our findings suggest that HHS may be an effective treatment for CRC cancer, and further studies are required to identify the active compounds in HHS.