Hepatoma cells are relatively resistant to TRAIL. We have previously shown that isoobtusilactone A (IOA), a potent anticancer agent isolated from Cinnamomum kotoense, induced mitochondria-mediated apoptosis in hepatoma cells. Here, we report that IOA could potentiate TRAIL-induced apoptosis in Hep G2 cells. The combined treatment with IOA and TRAIL significantly induced caspase-dependent apoptosis. This correlated with the up-regulation of C/EBP homologous protein (CHOP) and death receptor 5 (DR5) protein levels. Gene silencing of the DR5 by small interfering RNA abrogated the apoptosis induced by the combined regimen of IOA and TRAIL, suggesting that the sensitization to TRAIL was mediated through DR5. By analyzing the DR5 promoter, we found that IOA induced a CHOP-dependent DR5 transactivation. DR5 expression after IOA treatment was accompanied by provoking intracellular reactive oxygen species (ROS) generation. Pretreatment with N-acetyl-L-cysteine (NAC) attenuated IOA-induced CHOP and DR5 expression and inhibited TRAIL-induced apoptosis. Taken together, our data suggested that ROS-dependent and CHOP-regulated DR5 expression played a pivotal role in the synergistic enhancement of TRAIL-induced apoptosis instigated by IOA in Hep G2 cells.