Glioblastoma (GBM) is the most malignant brain tumor and incurable. Medicarpin (MED), a flavonoid compound from the legume family, has multiple targets and anticancer properties. However, the role of MED in GBM remains unclear. The objective of this study was to explore the effects of MED on the apoptosis of GBM and to explain the potential molecular mechanisms. We found that the IC50 values of U251 and U‐87 MG cells treated with MED for 24 h were 271 μg/mL and 175 μg/mL, and the IC50 values for 48 h were 154 μg/mL and 161 μg/mL, respectively. Additionally, the cell cycle of U251 and U‐87 MG cells were arrested at the G2/M phase. Furthermore, the apoptosis rate of U251 and U‐87 MG cells increased from 6.26% to 18.36% and 12.46% to 31.33% for 48 h, respectively. The migration rate of U251 and U‐87 MG decreased from 20% to 5% and 25% to 15% for 12 h and these of U251 and U‐87 MG decreased from 50% to 28% and 60% to 25% for 24 h. MED suppressed GBM tumorigenesis, and improved survival rate of tumor‐bearing mice. Taken together, MED triggered GBM apoptosis through upregulation of pro‐apoptotic proteins (BID, BAX, CASP3, CASP8, and CYCS), showed strong inhibitory effects on cell proliferation and cell migration, and displayed anti‐tumor activity in nude mice.