Glioblastoma (GBM) exhibits significant intratumor heterogeneity (ITH), indicating the presence of tumor cells with diverse growth rates. Here, we aimed to identify fast-growing cells in GBM and elucidate the underlying mechanisms. Precisely targeting these cells could offer an improved treatment option. Our results found that targeting ALKBH5 expression impaired GBM proliferation and tumor stemness. Nuclear but not overall expression of ALKBH5 differs between monoclonal cells derived from the same patient with different proliferation rates. Mechanistically, ALKBH5 interacted with TAR DNA-binding protein 43 (TDP-43) in fast-growing cells. Furthermore, TDP-43 facilitated the nuclear localization of ALKBH5 and its binding to cell division cycle 25A (CDC25A) pre-mRNA. The TDP-43/ALKBH5 complex regulates CDC25A mRNA splicing via N6-methyladenosine (m