The authors previously reported that costunolide, an active compound isolated from the stem bark of Magnolia sieboldii, induced apoptosis via reactive oxygen species (ROS) and Bcl-2-dependent mitochondrial permeability transition in human leukemia cells. In the present study, the authors investigated whether mitogen-activated protein kinases (MAPKs) are involved in the costunolide-induced apoptosis in human promonocytic leukemia U937 cells. Treatment with costunolide resulted in the significant activation of c-Jun N-terminal kinase (JNK), but not of extracellular-signal-related kinase (ERK1/2) or p38. In vitro kinase assays showed that JNK activity was low in untreated cells but increased dramatically after 30 min of costunolide treatment. U937 cells co-treated with costunolide and sorbitol, a JNK activator, exhibited higher levels of cell death. In addition, inhibition of the JNK pathway using a dominant-negative mutation of c-jun and JNK inhibitor SP600125, significantly prevented costunolide-induced apoptosis. Furthermore, pretreatment with the antioxidant NAC (N-acetyl-L-cysteine) blocked the costunolide-stimulated activation of JNK while the overexpression of Bcl-2 failed to reverse JNK activation. Pretreatment with SP600125 recovered the costunolide-suppressed Bcl-2 expression. These results indicate that costunolide-induced JNK activation acts downstream of ROS but upstream of Bcl-2, and suggest that ROS-mediated JNK activation plays a key role in costunolide-induced apoptosis. Moreover, the administration of costunolide (intraperitoneally once a day for 7 d) significantly suppressed tumor growth and increased survival in 3LL Lewis lung carcinoma-bearing model.