Pasireotide is a new generation somatostatin analogue used for the treatment of acromegaly, Cushing disease, and neuroendocrine tumors. As side effect, it may reduce the secretion of insulin, GLP-1, and GIP, causing hyperglycemia in some patients. Of note, the diabetogenic effects of pasireotide are reduced in vivo by co-administration of liraglutide or vildagliptin, two incretin-based drugs. The objective of this work was to evaluate the direct effects of pasireotide on pancreatic β-cell function and survival, the ability of different incretin-based drugs to prevent pasireotide action, and the molecular mechanisms underlying these events. To this aim, INS-1E cells and human pancreatic islets were stimulated with pasireotide or DMSO as a control; in another set of experiments, INS-1E cells were pretreated with a GLP-1 receptor agonist (GLP-1RA, i.e., exendin-4, lixisenatide, or liraglutide) prior to stimulation with pasireotide. Glucose-stimulated insulin secretion (GSIS), insulin content, and apoptosis levels were assessed through specific ELISA assays, while signaling proteins and calcium levels were evaluated by immunoblotting and fluorimetric assay, respectively. We found that pasireotide administration induced apoptosis and reduced GSIS, without altering insulin content, in both INS-1E cells and human islets. All GLP-1RAs tested prevented pasireotide-induced apoptosis in INS-1E cells, however the major pathways involved in β-cell apoptosis, such as p38 MAPK, JNK1/2, CHOP, p53, and ERK1/2, were not found to be activated by pasireotide, thus the molecular mechanism implicated was not clarified. Furthermore, although AKT activation was found to be reduced by pasireotide and restored by GLP-1RAs, experiments conducted with a chemical inhibitor of AKT excluded its involvement in the preventive action of GLP-1RAs on pasireotide-induced apoptosis. On the other hand, GLP-1RAs (exendin-4 and liraglutide) prevented pasireotide-induced secretory dysfunction in INS-1E cells, by interfering with PKA and cytoplasmic calcium pathways. In conclusion, pasireotide reduces β-cell survival and GSIS, possibly contributing to the onset of hyperglycemia in some patients, and GLP-1RAs can prevent these deleterious effects. These results obtained in vitro and ex vivo support the co-administration of incretin-based drugs and pasireotide, whose therapeutic efficacy has already been demonstrated in vivo. Supported by Novartis and Fondazione Cianciola.