The central nervous system has long been thought to regulate insulin secretion, an essential process in the maintenance of blood glucose levels. However, the anatomical and functional connections between the brain and insulin-producing pancreatic β cells remain undefined. Here, we describe a functional transneuronal circuit connecting the hypothalamus to β cells in mice. This circuit originates from a subpopulation of oxytocin neurons in the paraventricular hypothalamic nucleus (PVN), and it reaches the islets of the endocrine pancreas via the sympathetic autonomic branch to innervate β cells. Stimulation of PVN neurons rapidly suppresses insulin secretion and causes hyperglycemia. Conversely, silencing of these neurons elevates insulin levels by dysregulating neuronal signaling and secretory pathways in β cells and induces hypoglycemia. PVN neuronal activity is triggered by glucoprivation. Our findings reveal that a subset of PVN neurons form functional multisynaptic circuits with β cells in mice to regulate insulin secretion, and their function is necessary for the β cell response to hypoglycemia.