Decellularized extracellular matrix (dECM) is a valuable tool for generating three-dimensional in vitro tumor models that effectively recapitulate tumor-extracellular matrix (ECM) interactions. However, in current culture models, the components and structures of dECM are enzymatically disrupted to form hydrogels, making it difficult to recapitulate the native ECM. Additionally, when studying ECM-cell interactions, large-volume tumor culture models are incompatible with traditional experimental techniques and the nutrient-oxygen concentration gradient, which is a significant confounding factor. To address these issues, we developed a decellularized brain extracellular matrix slice (dBECMS) glioblastoma (GBM) culture model. This model possesses good light transmittance and substance diffusivity, making it compatible with traditional experimental techniques without forming nutrient-oxygen concentration gradients. Through transcriptomic analysis, we found that native brain ECM has a broad impact on glioma cells; the impact involves the ECM-ECM receptor interactions and the ECM and metabolic reprogramming. Further experiments demonstrated that dBECMS promoted glucose consumption and lactate production in GBM cells. Silver staining experiments revealed abundant proteins in the media of dBECMS, suggesting the degradation of the brain ECM by GBM cells. Transcriptome analysis also showed that the dBECMS-GBM culture model more accurately recapitulated the transcriptional profile of GBM than the two-dimensional culture. We experimentally demonstrated that the dBECMS-GBM model enhanced the resistance of GBM cells to temozolomide and increased the stemness of GBM cells. Additionally, we demonstrated the feasibility of the dBECMS-GBM model as a platform for drug response modeling. STATEMENT OF SIGNIFICANCE: The decellularized brain extracellular matrix (ECM) slice glioblastoma culture model mimics the interaction between native brain ECM and glioblastoma when glioblastoma infiltrates the brain and reveals the effects of native brain ECM on glioblastoma metabolism, ECM reprogramming, drug responsiveness, and stemness.Copyright © 2022. Published by Elsevier Ltd.