Mitochondrial GTP (mtGTP)-insensitive mutations in glutamate dehydrogenase (GDH(H454Y)) result in fasting and amino acid-induced hypoglycemia in Hyperinsulinemia Hyperammonemia (HI/HA). Surprisingly, hypoglycemia may occur in this disorder despite appropriately suppressed insulin. To better understand the islet-specific contribution transgenic mice expressing the human activating mutation in beta-cells (H454Y mice) were characterized in vivo. As in the humans with HI/HA, H454Y mice had fasting hypoglycemia but plasma insulin concentrations were similar to the controls. Paradoxically, both glucose- and glutamine-stimulated insulin secretion were severely impaired in H454Y mice. Instead, lack of a glucagon response during hypoglycemic clamps identified impaired counter regulation. Moreover, both insulin and glucagon secretion were impaired in perifused islets. Acute pharmacologic inhibition of GDH restored both insulin and glucagon secretion and normalized glucose tolerance in vivo. These studies support the presence of a mtGTP-dependent signal generated via beta-cell GDH that inhibits alpha-cells. As such, in children with activating GDH mutations of HI/HA this insulin-independent glucagon suppression may contribute importantly to symptomatic hypoglycemia. The identification of a human mutation causing congenital hypoglucagonemic hypoglycemia highlights a central role of the mtGTP-GDH-glucagon axis in glucose homeostasis.