Here, we demonstrate that the voltage-gated proton channel Hv1 represents a regulatory mechanism for insulin secretion of pancreatic islet β cell and glucose homeostasis. In vivo, Hv1-deﬁcient mice display hypoinsulinaemia, hyperglycemia and glucose intolerance due to reduced insulin secretion, but normal peripheral insulin sensitivity. In vitro, islets from Hv1-deﬁcient and heterozygous mice, β cells and islets with siRNA-mediated knockdown of Hv1 exhibit a marked defect in secretagogue-induced insulin secretion. A decrease in α and β cell masses has been observed in islets from Hv1-deficient mice. Furthermore, Hv1-deficient β cell has an impairment on glucose- and sulfonylurea-induced intracellular Ca2+ homeostasis and membrane depolarization. Collectively, our in vitro and in vivo results indicate that Hv1 is required for insulin secretion in the β cell and that dysfunction of Hv1 may contribute to the pathophysiology of type 2 diabetes. Our study sheds light on a new biological function of the proton channel.