Regulation of reproduction and energy homeostasis are linked, but our understanding of the central neural mechanisms subserving this connection is incomplete. Gonadotropin-inhibiting hormone (GnIH) is a neuropeptide that negatively regulates reproduction and stimulates food intake. Neuropeptide Y (NPY) and products of the pro-opiomelanocortin (POMC) precursor (beta-endorphin melanocortins) are appetite regulating peptides produced in the neurons of the arcuate nucleus; these peptides also regulate reproduction. In this study we determined the effects of GnIH on NPY and POMC neurons. Using brain slices from mice with transgenes for fluorescent tags in the two types of neuron and patch clamp electrophysiology, a predominant inhibitory effect of GnIH was observed. GnIH (100 nM) inhibited the firing rate in POMC cells, confirming results of others and consistent with the stimulatory effect of GnIH on food intake. Paradoxically (since both GnIH and NPY stimulate food intake), GnIH also had a predominantly inhibitory effect on action potential activity in NPY cells. GnIH also inhibited secretion of NPY and alpha-melanocyte stimulating hormone secretion in incubated hypothalamic blocks. GnIH (100 ng) injected into the cerebral ventricles of mice did not increase the number of NPY cells that were positively immunostained for c-Fos. Finally, dual label immunocytochemistry showed that 20 of NPY neurons had close contacts from GnIH fibres/varicosities. In conclusion, we confirm a negative effect of GnIH on POMC cells and demonstrate a paradoxical reduction of electrophysiological and functional activity in NPY cells. This article is protected by copyright. All rights reserved.