A hallmark of diabetes is an absolute or relative reduction in the number of functional β cells. Therapies that could increase the number of endogenous β cells under diabetic conditions would be desirable. Prevalent gene targeting mouse models for assessing β-cell proliferation and diabetes pathogenesis only address whether deletion of a gene prevents the development of diabetes. Models testing whether acute excision of a single gene can ameliorate or reverse preexisting hyperglycemia in established diabetes remain to be explored, which could directly validate the effect of gene excision on treating diabetes. Here, we report that acute and temporally controlled excision of the Men1 gene, which encodes menin, ameliorated preexisting hyperglycemia in streptozotocin-treated mice. Moreover, Men1 excision also improved the preexisting hyperglycemia and glucose intolerance in genetic db/db diabetic mice. Furthermore, acute Men1 excision reversed preexisting glucose intolerance in high-fat diet-fed mice. Men1 excision improved glucose metabolism at least partly through increasing proliferation of endogenous β cells and islet size. Acute Men1 excision up-regulated a group of proproliferative genes in pancreatic islets. Together, these findings demonstrate that established hyperglycemia can be reversed through repression of a single gene, Men1, in diabetic conditions, and suggest that menin is a vital regulator in pathogenesis of diabetes.