Mitochondrial glucose metabolism is critical for glucose-stimulated insulin secretion and glucose homeostasis in pancreatic beta cells. We previously showed that KCNH6, a voltage-dependent potassium (Kv) channel, participated regulation of insulin secretion in pancreatic beta cells, however, its role in mitochondrial metabolism remains unclear. Since we recently found that KCNH6 distributed in mitochondria, in this study, we investigated the role of KCNH6 in regulating mitochondrial function in pancreatic beta cells by using a beta cell-specific knockout (KCNH6-beta KO) mouse model. Proteomics analysis of islets indicated that multiple proteins involved in mitochondrial metabolism were dysregulated in islets of KCNH6-beta KO mice. Additionally, KCNH6-deficient beta cells exhibited damaged mitochondria morphology and oxidative respiration dysfunction, which manifested as decreased glucose-induced ATP production, elevated NADH/NAD+ ratio and ROS levels. Impaired mitochondrial metabolism in beta KO islets were significantly alleviated after the re-expression of KCNH6. Mechanistically, a physical interaction between KCNH6 and complex I assembly subunit Ndufa13 was detected, providing direct evidence of KCNH6's ability to regulate mitochondrial function. These results suggested that KCNH6 could be a promising therapeutic target for improving energy metabolism in beta cells.