Background : Ginseng, one of most famous traditional oriental medicines, has been known for a number of pharmacological properties including anti-tumor, anti-diabetic, anti-fatigue, anti-stress, anti-oxidative, and anti-aging effects. 20(S)-Protopanaxadiol (PPD), a intestinal metabolite of ginsenosides, is one of the active ingredients in ginseng. In this study, we investigated the synergistic anticancer effect of 20(S)-PPD and temozolomide (TMZ) and the mechanism of 20(S)-PPD on glioblastoma cells. Methods and Results : We examined cell viability and the morphological changes of C6 cells after treatment of 20(S)-PPD and TMZ. 20(S)-PPD showed a potent antiproliferative activity against C6 cells by triggering apoptosis. 20(S)-PPD-induced apoptosis was characterized by a dose-dependent mitochondrial damage. 20(S)-PPD and TMZ had a synergistic effect in increasing mitochondrial damage via caspase 3 activation. Conclusion : These results revealed an unexpected mechanism of 20(S)-PPD and TMZ, triggering a mitochondrial-mediated apoptosis in C6 cells. Our findings encourage further studies of 20(S)-PPD as a promising chemopreventive agent against glioblastoma.