Glucagon-like peptide-1 (GLP-1) is a potent regulator of glucose-stimulated insulin secretion whose mechanisms of action are only partly understood. In the present paper, we show that at low (3mM) glucose concentrations, GLP-1 increases the free intramitochondrial concentrations of both Ca2+ ([Ca2+]m), and ATP ([ATP]m) in clonal MIN6 β-cells. Suggesting that cAMP-mediated release of Ca2+ from intracellular stores is responsible for these effects, increases in [ATP]m that were induced by GLP-1 were completely blocked by the Rp isomer of adenosine-3′,5′-cyclic monophosphothioate (Rp-cAMPS), or by chelation of intracellular Ca2+. Furthermore, inhibition of Ins(1,4,5)P3 (IP3) receptors with xestospongin C, or application of ryanodine, partially inhibited GLP-1-induced [ATP]m increases, and the simultaneous blockade of both IP3 and ryanodine receptors (RyR) completely eliminated the rise in [ATP]m. GLP-1 appeared to prompt Ca2+-induced Ca2+ release through IP3 receptors via a protein kinase A (PKA)-mediated phosphorylation event, since ryanodine-insensitive [ATP]m increases were abrogated with the PKA inhibitor, H89. In contrast, the effects of GLP-1 on RyR-mediated [ATP]m increases were apparently mediated by the cAMP-regulated guanine nucleotide exchange factor cAMP-GEFII, since xestospongin C-insensitive [ATP]m increases were blocked by a dominant-negative form of cAMP-GEFII (G114E,G422D). Taken together, these results demonstrate that GLP-1 potentiates glucose-stimulated insulin release in part via the mobilization of intracellular Ca2+, and the stimulation of mitochondrial ATP synthesis.