Glucose-responsive (GR) neurons in the hypothalamus are thought to be critical in glucose homeostasis, but it is not known how they function in this context. Kir6. 2 is the pore-forming subunit of K ATP channels in many cell types, including pancreatic β-cells and heart. Here we show the complete absence of both functional ATP-sensitive K+(K ATP) channels and glucose responsiveness in the neurons of the ventromedial hypothalamus (VMH) in Kir6. 2−/− mice. Although pancreatic α-cells were functional in Kir6. 2−/−, the mice exhibited a severe defect in glucagon secretion in response to systemic hypoglycemia. In addition, they showed a complete loss of glucagon secretion, together with reduced food intake in response to neuroglycopenia. Thus, our results demonstrate that K ATP channels are important in glucose sensing in VMH GR neurons, and are essential for the maintenance of glucose homeostasis.