Although the patients with t(8;21) acute myeloid leukemia (AML) have a favorable prognosis compared with other non-acute promyelocytic leukemia AML patients, only similar to 50% patients with this relatively favorable subtype can survive for 5 years and refractory/relapse is common in clinical practice. So it is necessary to find novel agents to treat this type of AML. In this study, the effects and the mechanisms of plumbagin and recombinant soluble tumor necrosis factor-alpha-related apoptosis-inducing ligand (rsTRAIL) on leukemic Kasumi-1 cells were primarily investigated. Plumbagin and/or rsTRAIL could significantly inhibit the growth of Kasumi-1 cells and induce apoptosis in vitro and in vivo. Plumbagin enhanced TRAIL-induced apoptosis of Kasumi-1 cells in association with mitochondria damage, caspase activation, upregulation of death receptors (DRs) and decreased cFLIP expression. The effects of plumbagin on the expression of DR5, Bax and cFLIP could be partially abolished by the reactive oxygen species (ROS) scavenger NAC. Glutathione (GSH) depletion by plumbagin increased the production of ROS. In vivo, there was no obvious toxic pathologic change in the heart, liver and kidney tissues in any of the groups. Comparing with the control mice, a significantly increased number of apoptotic cells were observed in the combined treated mice by flow cytometry. Plumbagin also increased the expression of DR4 and DR5 in cells of xenograft tumors. Collectively, our results suggest that both plumbagin and rsTRAIL could be used as a single agent or synergistical agents to induce apoptosis of leukemic Kasumi-1 cells in vitro and in vivo.