Although the N6-methyladenine (N6-mA) DNA modification has been shown to exist within human genomic DNA, its biological function remains unclear. To investigate the role of N6-mA in glioblastoma, Xie et al. first showed that N6-mA was upregulated in human patient-derived glioblastoma stem cells (GSCs) and primary human tumor specimens. Genomic profiling revealed that N6-mA is distributed in intergenic regions and overlaps with heterochromatic histone modifications. Deletion of the N6-mA demethylase ALKBH1 increased N6-mA levels and decreased chromatin accessibility in GSCs. Sites with increased N6-mA modifications were found in ALKBH1-regulated genes associated with K3K9me3-containing heterochromatin regions, suggesting that ALKBH1 depletion promotes heterochromatin formation by increasing N6-mA levels to repress gene expression. Silencing ALKBH1 inhibited proliferation and the self-renewal ability of GSCs, as well as tumor growth in mice xenograft models. This study revealed the relationship between N6-mA and chromatin status and provided a potential therapeutic target for glioblastoma therapy.