Glioma is one of the most lethal malignancies and molecular regulators driving gliomagenesis are incompletely understood. Although temozolomide (TMZ) has been applied for malignant gliomas as a canonical chemotherapy, the treatment of glioma still remains limited due to frequently developed resistance to TMZ. Therefore, promising strategies that sensitize glioma cells to temozolomide are overwhelming to develop. Here we found that the expression of dihydrofolate reductase (DHFR) and thymidylate synthetase (TYMS), which played an essential role in folate metabolism and several types of tumors, were up-regulated in both human glioma tissues and cell lines, and overexpression of DHFR/TYMS promoted the proliferation of glioma cells. Notably, inhibition of DHFR/TYMS by pemetrexed exhibited synergistic anti-glioma activity with TMZ in both cell lines and U251 xenografts, which suggested potential combined chemotherapy for glioma. Mechanistically, the synergistic effect of inhibition of DHFR/TYMS with TMZ was due to activated AMPK and subsequently suppressed mTOR signaling pathway. Taken together, these findings identify an uncharacterized role of DHFR/TYMS in glioma growth and TMZ sensitivity mediated by AMPK-mTOR signal pathway, and provide a prospective approach for improving the anti-tumor activity of TMZ in glioma.