Recent success with the use of glucagon-like peptide-1 (GLP-1) receptor analogs and dipeptidyl peptidase-4 inhibitors for the treatment of patients with diabetes has highlighted the role of the intestine as an endocrine organ. Gut-derived hormones, including glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, and ghrelin, have important roles in the control of energy metabolism and food intake, and are associated with the metabolic syndrome. In this study, we isolated and identified a new intestine-derived hormone, betagenin, and showed that it stimulates insulin secretion and β-cell proliferation and suppresses β-cell apoptosis. Adenovirus-mediated expression of betagenin restored the blood glucose concentrations and hemoglobin A1c (HbA1c) levels of mice with streptozotocin-induced diabetes to normal and increased their β-cell mass. Transgenic mice overexpressing betagenin exhibited more than three-fold higher β-cell mass than WT mass, whereas that of KO mice was four-fold lower. A synthetic peptide representing the sequence of purified and secreted betagenin enhanced glucose-dependent insulin secretion in human and mouse pancreatic islets and stimulated the proliferation of the pancreatic β-cell line MIN6 through extracellular signal-regulated kinase 1/2-dependent signaling. The intravenous administration of this peptide to streptozotocin mice stimulated the proliferation of pancreatic β-cells in vivo, and the intraperitoneal administration of betagenin ameliorated diabetes and restored β-cell mass. These results indicate that betagenin may reduce blood glucose concentration and induce β-cell regeneration in patients with diabetes.