High glucose (HG)-induced oxidative stress contributes to the dysfunction of pancreatic beta cells in diabetes. The voltage-gated proton channel Hv1 has been proposed to support reactive oxygen species (ROS) production during respiratory bursts. However, the effect of Hv1 on glucotoxicity in pancreatic beta cells is not clear yet. In this study, we examined the protective effects of Hv1-deficiency in HG cultured cells. Following 48 h of treatment with 30 mM high glucose, Hv1 KO beta cells showed higher cell viability, lower cell apoptosis and a more stable insulin gene expression level compared to WT cells. In both control and HG cultured beta cells, deficiency of Hv1 decreased the glucose- and PMA-induced ROS production. Finally, HG incubation led to NOX4 upregulation in WT beta cells, which could be inhibited by HV1 deficiency. In conclusion, Hv1-deficiency prevents the HG treatment-induced NOX4 upregulation and protects beta cells from glucotoxicity. (C) 2019 Published by Elsevier Inc.