Introduction: The aim of the study is to investigate whether acute or chronic central administration of the hypothalamic neuropeptide 26RFa may ameliorate the glycemic control of obese/diabetic mice. Methods: Mice were treated for 4 months with a high-fat (HF) diet and received a single i.c.v. injection of 26RFa (3 µg) or a chronic i.c.v. administration of the peptide during 28 days via osmotic minipumps (25 µg/day). i.p. and oral glucose (GLU) tolerance tests, insulin (INS) tolerance test, glucose-stimulated insulin secretion (GSIS), food/water intake, horizontal/vertical activity, energy expenditure, meal pattern, and whole-body composition were monitored. In addition, 26RFa and GPR103 mRNA expressions as well as plasma 26RFa levels were evaluated by RT-QPCR and radioimmunoassay. Results: Acute administration of 26RFa in HF mice induced a robust antihyperglycemic effect by enhancing INS secretion, whereas chronic administration of the neuropeptide is unable to improve glucose homeostasis in these obese/diabetogenic conditions. By contrast, chronic 26RFa treatment induced an increase of the body weight accompanied with an enhanced food intake and a decreased energy expenditure. Finally, we show that the HF diet does not alter the hypothalamic expression of the 26RFa/GPR103 neuropeptidergic system nor the levels of circulating 26RFa. Conclusion: Our data indicate that the central beneficial effect of 26RFa on glucose homeostasis, by potentiating GSIS, is preserved in HF mice. However, chronic administration of the neuropeptide is unable to balance glycemia in these pathophysiological conditions, suggesting that the hypothalamic 26RFa/GPR103 neuropeptidergic system mainly affects short-term regulation of glucose metabolism.