Chaperone-mediated autophagy (CMA) is a homeostatic process essential for the lysosomal degradation of a selected subset of the proteome. CMA activity directly depends on the levels of LAMP2A, critical receptor for CMA substrate proteins at the lysosomal membrane. In glioblastoma (GBM), the most common and aggressive brain cancer in the adulthood, high levels of LAMP2A in the tumor and tumor-associated pericytes have been linked to temozolomide resistance. However, the implication of LAMP2A, and hence CMA, in any cancer stem cell type, and specifically, in glioblastoma stem cells (GSCs) remains unknown. In this work, we show that LAMP2A expression is enriched in patient-derived GSC population and its knock-down diminishes GSCs tumorigenic activities. Proteomic and transcriptomic analysis of LAMP2A knocked-down GSCs revealed reduced extracellular matrix (ECM) interaction effectors in both analyses. Moreover, mitochondrial metabolism related pathways and immune system were differentially deregulated at proteome level. Furthermore, clinical samples of GBM tissue presented overexpression of LAMP2 and its high levels correlated with advanced glioma grade and poor overall survival. In conclusion, we identified a novel role of CMA directly regulating GSCs activity via multiple pathways at proteome and transcriptome level.