OBJECTIVES: Glioblastoma (GB) is a grade IV glial tumor characterized by high malignancy and dismal prognosis, primarily due to high recurrence rates and therapeutic resistance. The epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK), regulates signaling pathways, including cell growth, proliferation, survival, migration, and cell death. Many cancers utilize immune checkpoints (ICs) to attenuate immune responses. CD73 is an enzyme that functions as an IC by hydrolyzing AMP to adenosine, suppressing immune cells in the tumor microenvironment. However, the role of CD73 in resistance to EGFR inhibitors is poorly understood. This study aims to elucidate the resistance mechanisms induced by anti-EGFR treatment and to evaluate an anti-CD73 approach to overcome resistance mediated by anti-EGFR monotherapy. METHODS: The U251 GB cell line was treated with AG1478, an EGFR inhibitor, and the resistance markers MRP-1, PD-L1, and CD73 were evaluated using flow cytometry. Additionally, we assessed the combination effects of AG1478 and APCP (an EGFR and a CD73 inhibitor, respectively) on cell cycle progression, proliferation, apoptosis, and migration . RESULTS: We observed high EGFR, PD-L1, and CD73 expression in human GB cells. The treatment with AG1478 increased the expression of resistance markers MRP-1, PD-L1, and CD73, whereas it decreased CTLA-4. The combination of AG1478 and APCP did not alter proliferation or apoptosis but interfered with cell cycling, arresting the cells in the G1 phase, decreasing cell motility and partially reversing MRP-1 overexpression. CONCLUSION: In summary, our findings indicate that CD73 inhibition has a modest effect in overcoming resistance to EGFR monotherapy . Thus, further studies are needed, as the inhibition of both EGFR and CD73 affects cells in the tumor microenvironment and could potentially enhance anti-tumor immunity.