PR/SET Domain 9 (PRDM9) is normally restricted to gametes, where it deposits H3K4me3 marks at sites of meiotic recombination. We discovered that PRDM9 is aberrantly re-expressed in glioblastoma, where it maintains drug tolerance to microtubule-targeting agent (MTA) chemotherapy. Using H3K4me3 ChIP-seq in glioblastoma stem cells, PRDM9 knockout models, and patient tumours, we confirmed PRDM9 activity and found that its inhibition causes widespread loss of promoter H3K4me3 marks. RNA-sequencing revealed that cholesterol biosynthesis genes are especially sensitive to PRDM9 inhibition, with H3K4me3 enrichment at promoters of key genes (e.g., DHCR24, DHCR7) strongly correlating with their transcript levels in tumours. PRDM9 inhibition or knockout lowered cholesterol and precursor metabolites, and supplementation with cholesterol restored drug tolerance in persisters. Mechanistically, PRDM9 inhibition increased lipid peroxidation in persisters, while N-acetylcysteine supplementation rescued persister survival. Together, these findings establish PRDM9-mediated H3K4me3 as a crucial regulator of cholesterol metabolism that supports glioblastoma persister survival during chemotherapy.