Glucose-sensing neurons in the brainstem participate in the regulation of energy homeostasis but have been poorly characterized because of the lack of specific markers to identify them. Here we show that GLUT2-expressing neurons of the nucleus of the tractus solitarius form a distinct population of hypoglycemia-activated neurons. Their response to low glucose is mediated by reduced intracellular glucose metabolism, increased AMP-activated protein kinase activity, and closure of leak K⁺ channels. These are GABAergic neurons that send projections to the vagal motor nucleus. Light-induced stimulation of channelrhodospin-expressing GLUT2 neurons in vivo led to increased parasympathetic nerve firing and glucagon secretion. Thus GLUT2 neurons of the nucleus tractus solitarius link hypoglycemia detection to counterregulatory response. These results may help identify the cause of hypoglycemia-associated autonomic failure, a major threat in the insulin treatment of diabetes.