The voltage-gated proton channel Hv1 is a potent acid extruder that participates in the extrusion of the intracellular acid. Here, we showed for the first time, Hv1 is highly expressed in mouse and human pancreatic islet beta-cells, as well as beta-cell lines. Imaging studies demonstrated that Hv1 resides in insulin-containing granules in beta-cells. Knockdown of Hv1 with RNA interference significantly reduces glucose and K-induced insulin secretion in isolated islets and INS-1 (832/13) beta-cells and has an impairment on glucose- and K+-induced intracellular Ca2+ homeostasis. Our data demonstrated that the expression of Hv1 in pancreatic islet beta-cells regulates insulin secretion through regulating Ca2+ homeostasis. (C) 2015 Elsevier Inc. All rights reserved.