<p>CD73 (ecto-5’-nucleotidase) is a metabolic immune checkpoint that dephosphorylates AMP to produce adenosine. Adenosine plays a pivotal role in immunosuppressive tumor microenvironment (TME) through adenosine receptors expressed on various immune cells. AB680, a specific CD73 inhibitor, is currently undergoing clinical trials for highly refractory cancers. In this study, we investigated the antitumor effects and mechanisms of AB680 in glioblastoma (GBM). By analyzing the expression pattern of CD73 across all cell types in orthotopic naïve G422^TN-GBM tumors (d 7), we found that CD73 and its associated adenosine metabolic signaling were significantly elevated in G422^TN-GBM cells compared to all other cell types. High CD73 expression was also observed in human GBM samples and was correlated with shorter patient survival. Administration of AB680 significantly prolonged survival in G422^TN-GBM-bearing mice, reduced tumor size, cell proliferation, angiogenesis, and enhanced microglia activation and anti-tumor immune responses. Metabolomic analysis revealed that AB680 markedly increased ADP and AMP levels in the TME of orthotopic G422^TN-GBM, thereby stimulating the activation of P2RY12⁺ microglia to exert their M1-like anti-cancer functions, as confirmed by human GBM scRNA-seq and G422^TN-GBM snRNA-seq data. Furthermore, AB680 combined with RT/TMZ exhibited synergistic therapeutic effects by reversing RT/TMZ-induced increases in adenosine levels and promoting the transformation of P2RY12⁺ microglia. Overall, this study demonstrates that targeting CD73 with AB680 alters purine metabolism in the GBM microenvironment, promotes the transformation of P2RY12⁺ microglia, and triggers robust anti-tumor immune responses. These results support the rationale for AB680-based therapeutic clinical trials for GBM.</p>