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Simple Summary: Tumor cells use autophagy as a pro-survival strategy. However, several studies have shown that excessive stimulation of the autophagic process can promote cell death; in this context, autophagy acquires an antitumor effect. To further explore the consequences of the cytotoxic effects induced by M2 muscarinic receptor activation that we have previously described both in glioblastoma multiforme (GBM) stable cell lines and in human GBM cancer stem cells, here we investigated the involvement of autophagy and apoptosis in the cell death engendered by treatment with M2 muscarinic agonists. Moreover, we compared the effects mediated by orthosteric and dualsteric M2 muscarinic agonists in the modulation of these different mechanisms of cell death. Background: Although autophagy is a pro-survival process of tumor cells, it can stimulate cell death in particular conditions and when differently regulated by specific signals. We previously demonstrated that the selective stimulation of the M2 muscarinic receptor subtype (mAChR) negatively controls cell proliferation and survival and causes oxidative stress and cytotoxic and genotoxic effects in both GBM cell lines and GBM stem cells (GSCs). In this work, we have evaluated whether autophagy was induced as a downstream mechanism of the observed cytotoxic processes induced by M2 mAChR activation by the orthosteric agonist APE or the dualsteric agonist N8-Iper (N8). Methods: To assess the activation of autophagy, we analyzed the expression of LC3B using Western blot analysis and in LC3B-EGFP transfected cell lines. Apoptosis was assessed by measuring the protein expression of Caspases 3 and 9. Results: Our data indicate that activation of M2 mAChR by N8 promotes autophagy in both U251 and GB7 cell lines as suggested by the LC3B-II expression level and analysis of the transfected cells by fluorescence microscopy. Autophagy induction by M2 mAChRs is regulated by the decreased activity of the PI3K/AKT/mTORC1 pathway and upregulated by pAMPK expression. Downstream of autophagy activation, an increase in apoptosis was also observed in both cell lines after treatment with the two M2 agonists. Conclusions: N8 treatment causes autophagy via pAMPK upregulation, followed by apoptosis in both investigated cell lines. In contrast, the absence of autophagy in APE-treated GSC cells seems to indicate that cell death could be triggered by mechanisms alternative to those observed for N8. [ABSTRACT FROM AUTHOR]