26RFa is a hypothalamic RFamide neuropeptide that was identified as the endogenous ligand of the orphan G protein-coupled receptor, GPR103, and that stimulates appetite in mice. Up until now, the mechanism of action of 26RFa in the hypothalamic control of food intake remains unknown. The high density of GPR103 in the arcuate nucleus (Arc) prompted us to investigate, in the present study, the effects of 26RFa on the rat neuropeptide Y (NPY)/proopiomelanocortin (POMC) system. Intracerebroventricular injection of 26RFa stimulated NPY expression and release in the basal hypothalamus, whereas it decreased POMC expression and alpha-MSH release, and these effects were associated with an increase in food intake. A double in situ hybridization procedure indicated that the 26RFa receptor is present in NPY neurons of the Arc, but not in POMC neurons. Central administration of NPY Y1 and Y5 receptor antagonists abolished the inhibitory effects of 26RFa on POMC expression and alpha-MSH release, and reversed 26RFa-induced food consumption. Finally, 26RFa antagonized the effects of leptin on NPY expression and release, POMC expression and alpha-MSH release, and food intake. Altogether, the present data demonstrate for the first time that 26RFa exerts its orexigenic activity by stimulating the release of NPY in the Arc, which in turn inhibits POMC neurons by activating the Y1 and Y5 receptors. It is also suggested that the balance 26RFa/leptin is an important parameter in the maintenance of energy homeostasis.