l-Arginine (l-Arg) is an insulin secretagogue, but the molecular mechanism whereby it stimulates insulin secretion from β-cells is not known. The possibility that l-Arg regulates insulin secretion through a G protein-coupled receptor (GPCR)-mediated mechanism is suggested by the high expression of the nutrient receptor GPCR family C group 6 member A (GPRC6A) in the pancreas and TC-6 β-cells and the finding that Gprc6a^−/]minus] mice have abnormalities in glucose homeostasis. To test the direct role of GPRC6A in regulating insulin secretion, we evaluated the response of pancreatic islets derived from Gprc6a^−/]minus] mice to l-Arg. We found that the islet size and insulin content were decreased in pancreatic islets from Gprac6a^−/]minus] mice. These alterations were selective for β-cells, because there were no abnormalities in serum glucagon levels or glucagon content of islets derived from Gprac6a^−/]minus] mice. Significant reduction was observed in both the pancreatic ERK response to l-Arg administration to Gprc6a^−/]minus] mice in vivo and l-Arg-induced insulin secretion and production ex vivo in islets isolated from Gprc6a^−/]minus] mice. l-Arg stimulation of cAMP accumulation in isolated islets isolated from Gprc6a^−/]minus] mice was also diminished. These findings suggest that l-Arg stimulation of insulin secretion in β-cells is mediated, at least in part, through GPRC6A activation of cAMP pathways.