Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with limited therapeutic options and poor clinical outcomes. In this study, we report the rational design and synthesis of a novel series of pyrimidine-4,6-diamine derivatives targeting OLIG2, a critical transcription factor involved in GBM progression. Among the synthesized compounds, B01 emerged as a potent and selective OLIG2 inhibitor, exhibiting strong anti-proliferative activity in U87 and U251 cells with IC50 values of 7.0 μM and 6.4 μM, respectively. Mechanistic studies confirmed that B01 directly and dose-dependently downregulated nuclear OLIG2 levels, with an IC50 of 0.88 μM. Notably, B01 demonstrated synergistic anti-tumor activity in combination with temozolomide (TMZ). In vivo, B01 reduced tumor volume by 46 % compared to control in a U87 xenograft model, with enhanced efficacy observed in combination therapy. Pharmacokinetic analysis revealed a favorable half-life (t1/2 = 3.3 h) and systemic exposure. This study identifies a novel and selective OLIG2 inhibitor, B01, with potent anti-GBM activity and therapeutic synergy with TMZ, making it a compelling candidate for further preclinical development.