Cordycepol C, a novel sesquiterpene isolated from the cultured mycelia of Cordyceps ophioglossoides, contains a hydroperoxy group and is cytotoxic to HepG2 cells. So far, no sesquiterpenes have been found in the genus Cordyceps and it would be interesting to investigate the antitumor efficacy as well as the mechanism of action of this unusual sesquiterpene. In this study, we showed that cordycepol C induced apoptosis of the HepG2 cells without affecting the normal liver cell line L-02. Cordycepol C caused poly(ADP-ribose)polymerase-1 (PARP-1) cleavage and triggered the loss of mitochondrial membrane potential (Δψm) in HepG2 cells in a time- and dose-dependent manner, resulting in the nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (Endo G). We also found that cordycepol C induced the expression of Bax protein, followed by its translocation from the cytosol to mitochondria in both wild type and p53 knockdown HepG2 cells. However, cordycepol C could not cause cleavages of procaspase-3, -8, and -9. Caspase activities were not increased and Z-VAD-fmk, a caspase inhibitor, could not prevent the apoptosis induced by cordycepol C. These findings indicate that cordycepol C induces caspase-independent apoptosis in HepG2 cells through a p53-independent and Bax-mediated mitochondrial pathway, leading to the nuclear translocation of AIF and Endo G. Our study provides the molecular mechanism by which cordycepol C induces apoptosis in hepatocellular carcinoma cells and indicates the potential use of cordycepol C as an antitumor agent.