Whole-body glucose homeostasis is coordinated through secretion of glucagon and insulin from pancreatic islets. When glucose is low, glucagon is released from α-cells to stimulate hepatic glucose production. However, the mechanisms that regulate glucagon secretion from pancreatic α-cells remain unclear. Here we show that in α-cells, the interaction between fatty acid oxidation and glucose metabolism controls glucagon secretion. The glucose-dependent inhibition of glucagon secretion relies on pyruvate dehydrogenase and carnitine palmitoyl transferase 1a activity and lowering of mitochondrial fatty acid oxidation by increases in glucose. This results in reduced intracellular ATP and leads to membrane repolarization and inhibition of glucagon secretion. These findings provide a new framework for the metabolic regulation of the α-cell, where regulation of fatty acid oxidation by glucose accounts for the stimulation and inhibition of glucagon secretion.