Glioblastoma (GBM) is the most lethal brain cancer, with GBM stem cells (GSCs) driving therapeutic resistance and recurrence. Targeting GSCs offers a promising strategy for preventing tumor relapse and improving outcomes. We identify SUV39H1, a histone-3, lysine-9 methyltransferase, as critical for GSC maintenance and GBM progression. SUV39H1 is upregulated in GBM compared with normal brain tissues, with single-cell RNA-seq showing its expression predominantly in GSCs due to super-enhancer-mediated activation. Knockdown of SUV39H1 in GSCs impaired their proliferation and stemness. Whole-cell RNA-seq analysis revealed that SUV39H1 regulates G2/M cell cycle progression, stem cell maintenance, and cell death pathways in GSCs. By integrating the RNA-seq data with ATAC-seq data, we further demonstrated that knockdown of SUV39H1 altered chromatin accessibility in key genes associated with these pathways. Chaetocin, an SUV39H1 inhibitor, mimics the effects of SUV39H1 knockdown, reducing GSC stemness and sensitizing cells to temozolomide, a standard GBM chemotherapy. In a patient-derived xenograft model, targeting SUV39H1 inhibits GSC-driven tumor growth. Clinically, high SUV39H1 expression correlates with poor glioma prognosis, supporting its relevance as a therapeutic target. This study identifies SUV39H1 as a crucial regulator of GSC maintenance and a promising therapeutic target to improve GBM treatment and patient outcomes.