Abstract Glioblastoma (GBM) remains a formidable challenge in neuro-oncology, characterized by heterogeneity and aggressive tumor growth. The identification of novel biomarkers and therapeutic targets is crucial for advancing promising therapies. This study profiled patient-derived tumor samples utilizing single-cell RNA sequencing and proteomics platforms to uncover the upregulation of glycoprotein non-metastatic melanoma protein B (GPNMB) in tumor cells, particularly in post-treatment recurrent tumor cells, as well as in tumor-associated macrophages (TAMs), which constitute a major population of immune cells in treatment-refractory recurrent GBM. By exploring its expression patterns against normal tissue specimens and utilizing a series of patient-derived xenograft and humanized mouse models, we explored GPNMB as a target for Chimeric Antigen Receptor T cells (CAR-Ts) as a monotherapy and in rationally designed combination treatment regimens. We also demonstrated the utility of the GPNMB CAR-T cell in eliminating TAMs, a major contributor to immunosuppression in solid tumors. Our study reveals the development of GPNMB-targeting therapies as part of a promising and effective combinatorial treatment strategy for GBM. Citation Format: Sheila K Singh, Neil Savage, Franz J Zemp, Vaseem Shaikh, Shan Grewal, Nicholas Mikolajewicz, Hinda Najem, Chitra Venugopal, Thomas Kislinger, Amy Heimberger, Douglas Mahoney, Jason Moffat. GPNMB CAR T cells target both Glioblastoma and its immunosuppressive niche to relieve immune evasion [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A015.