Epithermal growth factor (EGF) regulates pancreatic β-cell survival, proliferation, and differentiation. EGF is synthesized in the pancreas and found in pancreatic juice, and diabetic animals have low levels of EGF. However, the role of EGF in regulating insulin secretion, the major function of pancreatic β-cells, has not been studied. Here we show that EGF rapidly and specifically stimulates insulin secretion in mouse pancreatic islets, as well as in a pancreatic β-cell line (MIN6 cells). These events were dependent on Ca2+ influx and phospholipase D (PLD) activity, particularly PLD2. In addition, EGF also increased plasma insulin levels and mediated glucose lowering in mice in vivo. Here, for the first time, we provide evidences that EGF as a novel secretagogue regulating plasma glucose levels and find the mechanism of EGF-induced insulin secretion.