Elevated hexosamine biosynthesis fuels tumor growth by facilitating protein and lipid glycosylation. But which enzyme in this pathway is better to serve as an antitumor target remains unclear. Here, we revealed that targeting GFAT1, the rate-limiting enzyme in hexosamine synthesis, exhibits limited inhibitory effects on glioblastoma (GBM), the most lethal brain tumor. This outcome is due to the compensation of NAGK-mediated hexosamine salvage pathway. Unexpectedly, inhibiting PGM3, which controls the flux of both de novo hexosamine synthesis and salvage pathways, down-regulates the expression of other enzymes in this pathway and suppresses SREBP-1, a critical lipogenic transcription factor, effectively inhibiting GBM growth. Unexpectedly, SREBP-1 transcriptionally up-regulates the expression of hexosamine synthesis enzymes, while inhibition of these enzymes in turn down-regulates SREBP-1 activation via reducing N-glycosylation of its transporter, SCAP. Our study identified PGM3 as a promising target for treating GBM. Its inhibition disrupts the SREBP-1 activation-hexosamine synthesis positive feedback regulation to effectively eliminate GBM cells.