Costunolide is an active sesquiterpene lactone isolated from the root of Saussurea lappa Clarke and is known to exhibit a variety of biological activities, including anti-carcinogenic and anti-inflammatory effects. Nevertheless, the pharmacological pathways of costunolide have not yet been fully elucidated. In this study, its cytotoxic effects were examined using AGS gastric cancer cells. Its treatment resulted in apoptosis in a dose- and time-dependent manner. The effects were attributed to the regulation of pro-apoptotic molecules and suppression of anti-apoptotic molecules. These results suggest that costunolide may be a candidate to deal with gastric cancers by chemopreventive agents.