Mutations in KCNH6 has been proved to cause hypoinsulinemia and diabetes in human and mice. Cisapride is a stomach-intestinal motility drug used to treat gastrointestinal dysfunction. Cisapride has been reported to be a potential inhibitor of the KCNH family, but it remained unclear whether cisapride inhibited KCNH6. Here, we discovered the role of cisapride on glucose metabolism, focusing on the KCNH6 potassium channel protein. Cisapride reduced blood glucose level and increased serum insulin secretion in wild-type (WT) mice fed standard normal chow/a high-fat diet or in db/db mice, especially when combined with tolbutamide. This effect was much stronger after 4 weeks of intraperitoneal injection. Whole-cell patch-clamp showed that cisapride inhibited KCNH6 currents in transfected HEK293 cells in a concentration-dependent manner. Cisapride induced an increased insulin secretion through the disruption of intracellular calcium homeostasis in a rat pancreatic beta-cell line, INS-1E. Further experiments revealed that cisapride did not decrease blood glucose or increase serum insulin in KCNH6 beta-cell knockout (Kcnh6-beta-KO) mice when compared with WT mice. Cisapride also ameliorated glucose-stimulated insulin secretion (GSIS) in response to high glucose in WT but not Kcnh6-beta-KO mice. Thus, our data reveal a novel way for the effect of KCNH6 in cisapride-induced hypoglycemia.