Simple SummaryAutophagy is a process in which the cell recycles components that are not needed at that moment and uses the resulting elements to satisfy more urgent needs. Depending on the specific context, this can be beneficial or detrimental for tumor development. We found that in glioblastoma, the most lethal brain tumor, autophagy is upregulated and contributes to glioblastoma stem cell survival under starvation. Importantly, the antitumor peptide TAT-Cx43266-283 blocks autophagy flux, contributing to the death of glioblastoma stem cells. This peptide induces glioblastoma stem cell death in nutrient-deprived and complete environments, while the effect of other unsuccessful drugs for glioblastoma depends on nutrient context, supporting the potential of TAT-Cx43266-283 as a treatment to improve the lives of glioblastoma patients.Autophagy is a physiological process by which various damaged or non-essential cytosolic components are recycled, contributing to cell survival under stress conditions. In cancer, autophagy can have antitumor or protumor effects depending on the developmental stage. Here, we use Western blotting, immunochemistry, and transmission electron microscopy to demonstrate that the antitumor peptide TAT-Cx43266-283, a c-Src inhibitor, blocks autophagic flux in glioblastoma stem cells (GSCs) under basal and nutrient-deprived conditions. Upon nutrient deprivation, GSCs acquired a dormant-like phenotype that was disrupted by inhibition of autophagy with TAT-Cx43266-283 or chloroquine (a classic autophagy inhibitor), leading to GSC death. Remarkably, dasatinib, a clinically available c-Src inhibitor, could not replicate TAT-Cx43266-283 effect on dormant GSCs, revealing for the first time the possible involvement of pathways other than c-Src in TAT-Cx43266-283 effect. TAT-Cx43266-283 exerts an antitumor effect both in nutrient-complete and nutrient-deprived environments, which constitutes an advantage over chloroquine and dasatinib, whose effects depend on nutrient environment. Finally, our analysis of the levels of autophagy-related proteins in healthy and glioma donors suggests that autophagy is upregulated in glioblastoma, further supporting the interest in inhibiting this process in the most aggressive brain tumor and the potential use of TAT-Cx43266-283 as a therapy for this type of cancer.