Significance of this studyWhat is already known about this subject? ► 26RFa is a biologically active peptide produced in abundance in the gut and the pancreas. ► 26RFa has been found to regulate glucose homeostasis by acting as an incretin and by increasing insulin sensitivity.What are the new findings?► Disruption of the 26RFa gene induces substantial alteration in the regulation of glucose homeostasis, with in particular a deficit in insulin production by the pancreatic islets, assessing therefore the notion that 26RFa is an important regulator of glucose homeostasis.How might these results change the focus of research or clinical practice?► Identification of a novel actor in the regulation of glucose homeostasis is crucial to better understand the general control of glucose metabolism in physiological and pathophysiological conditions, and opens new fields of investigation to develop innovative drugs to treat diabetes mellitus. AbStrActIntroduction 26RFa (pyroglutamyl RFamide peptide (QRFP)) is a biologically active peptide that has been found to control feeding behavior by stimulating food intake, and to regulate glucose homeostasis by acting as an incretin.The aim of the present study was thus to investigate the impact of 26RFa gene knockout on the regulation of energy and glucose metabolism.Research design and methods 26RFa mutant mice were generated by homologous recombination, in which the entire coding region of prepro26RFa was replaced by the iCre sequence. Energy and glucose metabolism was evaluated through measurement of complementary parameters. Morphological and physiological alterations of the pancreatic islets were also investigated.Results Our data do not reveal significant alteration of energy metabolism in the 26RFa-deficient mice except the occurrence of an increased basal metabolic rate. By contrast, 26RFa mutant mice exhibited an altered glycemic phenotype with an increased hyperglycemia after a glucose challenge associated with an impaired insulin production, and an elevated hepatic glucose production. Two-dimensional and three-dimensional immunohistochemical experiments indicate that the insulin content of pancreatic β cells is much lower in the 26RFa −/− mice as compared with the wild-type littermates.Conclusion Disruption of the 26RFa gene induces substantial alteration in the regulation of glucose homeostasis, with in particular a deficit in insulin production by the pancreatic islets. These findings further support the notion that 26RFa is an important regulator of glucose homeostasis.