Mammalian target of rapamycin (mTOR) is a protein kinase that integrates signals from mitogens and the nutrients, glucose and amino acids, to regulate cellular growth and proliferation. Previous findings demonstrated that glucose robustly activates mTOR in an amino acid-dependent manner in rodent and human islets. Furthermore, activation of mTOR by glucose significantly increases rodent islet DNA synthesis that is abolished by rapamycin. Glucagon-like peptide-1 (GLP-1) agonists, through the production of cAMP, have been shown to enhance glucose-dependent proinsulin biosynthesis and secretion and to stimulate cellular growth and proliferation. The objective of this study was to determine if the glucose-dependent and cAMP-mediated mechanism by which GLP-1 agonists enhance β-cell growth and proliferation is mediated, in part, through mTOR. Our studies demonstrated that forskolin-generated cAMP resulted in activation of mTOR at basal glucose concentrations as assessed by phosphorylation of S6K1, a downstream effector of mTOR. Conversely, an adenylyl cyclase inhibitor partially blocked glucose-induced S6K1 phosphorylation. Furthermore, the GLP-1 receptor agonist, Exenatide, dose-dependently enhanced phosphorylation of S6K1 at an intermediate glucose concentration (8 mmol/l) in a rapamycin-sensitive manner. To determine the mechanism responsible for this potentiation of mTOR, the effects of intra- and extracellular Ca²⁺ were examined. Glyburide, an inhibitor of ATP-sensitive K⁺ channels (K_ATP channels), provided partial activation of mTOR at basal glucose concentrations due to the influx of extracellular Ca²⁺, and diazoxide, an activator of K_ATP channels, resulted in partial inhibition of S6K1 phosphorylation by 20 mmol/l glucose. Furthermore, Exenatide or forskolin reversed the inhibition by diazoxide, probably through mobilization of intracellular Ca²⁺ stores by cAMP. BAPTA, a chelator of intracellular Ca²⁺, resulted in inhibition of glucose-stimulated S6K1 phosphorylation due to a reduction in cytosolic Ca²⁺ concentrations. Selective blockade of glucose-stimulated Ca²⁺ influx unmasked a protein kinase A (PKA)-sensitive component involved in the mobilization of intracellular Ca²⁺ stores, as revealed with the PKA inhibitor H-89. Overall, these studies support our hypothesis that incretin-derived cAMP participates in the metabolic activation of mTOR by mobilizing intracellular Ca²⁺ stores that upregulate mitochondrial dehydrogenases and result in enhanced ATP production. ATP can then modulate K_ATP channels, serve as a substrate for adenylyl cyclase, and possibly directly regulate mTOR activation.