Costunolide (C(15)H(20)O(2)) is a sesquiterpene lactone that was isolated from many herbal medicines and it has diverse effects according to previous reports. However, the anti-cancer effects and the mechanism of actions are still unknown in breast cancer. In this study, we first observed that costunolide inhibits cell growth in a dose-and time-dependent manner. To examine the mechanism by which costunolide inhibits cell growth, we checked the effect of costunolide on apoptosis and the cell cycle. Costunolide induced apoptosis through the extrinsic pathway, including the activation of Fas, caspase-8, caspase-3, and degradation of PARP. However, did not have the same effect on the intrinsic pathway as revealed by analysis of mitochondrial membrane potential (Δψm) with JC-1 dye and expression of Bcl2 and Bax proteins level. Furthermore, costunolide induced cell cycle arrest in the G2/M phase via decrease in Cdc2, cyclin B1 and increase in p21WAF1 expression, independent of p53 pathway in p53-mutant MDA-MB-231 cells and increases Cdc2-p21WAF1 binding. In addition, costunolide had a slight induced effect on ROS generation. Among the mechanisms of p21WAF1 induction examined, costunolide-induced increase in p21WAF1 expression was related with protein stability and ROS generation. Through this study we confirm that costunolide induces G2/M cell cycle arrest and apoptotic cell death via extrinsic pathway in MDA-MB-231 cells suggesting that it could be a promising anticancer drug especially for ER-negative breast cancer.