Cortex periplocae is the dried root bark of Periploca sepium Bge., a traditional Chinese herb medicine. It contains high amounts of cardiac glycosides. Several cardiac glycosides have been reported to inhibit tumor growth or induce tumor cell apoptosis. We extracted and purified cortex periplocae and identified periplocin as the active ingredient that inhibited the growth of TNF-related apoptosis-inducing ligand-(TRAIL-) resistant hepatocellular carcinoma cells. The antitumor activity of periplocin was further increased by TRAIL cotreatment. Periplocin sensitized TRAIL-resistant HCC through the following two mechanisms. First, periplocin induced the expression of DR4 and FADD. Second, the cotreatment of TRAIL and periplocin suppressed several inhibitors of apoptosis (IAPs). Both mechanisms resulted in the activation of caspase 3, 8, and 9 and led to cell apoptosis. In addition, intraperitoneal injection (IP) of periplocin repressed the growth of hepatocellular carcinoma (HCC) in xenograft tumor model in mice. In summary, periplocin sensitized TRAIL-resistant HCC cells to TRAIL treatment and resulted in tumor cell apoptosis and the repression of tumor growth in vivo.