Shikonin has been demonstrated to exhibit anti-cancer activity, but the underlying mechanisms are poorly understood. In this report, we showed that the administration of shikonin could result in the induction of apoptotic cell death of human hepatoma cell line, SK-Hep-1. As evident by the flow-cytometric studies, shikonin has the capability of generating increased amounts of intracellular reactive oxygen species (ROS) during the early stage of this apoptotic process (ca. one-hour), and subsequently accompanied by the dissipation of mitochondrial transmembrane potential (deltapsi (m)) at 3 hours. Further studies indicated that this apoptotic process could effectively be protected by the pretreatment of shikonin-treated cells with glutathione (GSH) and N-acetylcysteine (NAC), a precursor of GSH, but not by cyclosporin A (CyA), an inhibitor of mitochondrial permeability transition (MPT) pore. These data further proved that ROS-mediated oxidative stress was the pivotal element involved in the induction of apoptosis of SK-Hep-1 cells. Taken together, we suggest that shikonin-induced apoptosis of SK-Hep-1 cells proceeds by an oxidative stress-mediated pathway.