Neuropeptide Y (NPY), a member of the pancreatic polypeptide family, is an orexigenic hormone. GnRH-1 neurons express NPY receptors. This suggests a direct link between metabolic function and reproduction. However, the effect of NPY on GnRH-1 cells has been variable, dependent on metabolic and reproductive status of the animal. This study circumvents these issues by examining the role of NPY on GnRH-1 neuronal activity in an explant model that is based on the extra-central nervous system origin of GnRH-1 neurons. These prenatal GnRH-1 neurons express many receptors found in GnRH-1 neurons in the brain and use similar transduction pathways. In addition, these GnRH-1 cells exhibit spontaneous and ligand-induced oscillations in intracellular calcium as well as pulsatile calcium-controlled GnRH-1 release. Single-cell PCR determined that prenatal GnRH-1 neurons express the G protein-coupled Y1 receptor (Y1R). To address the influence of NPY on GnRH-1 neuronal activity, calcium imaging was used to monitor individual and population dynamics. NPY treatment, mimicked with Y1R agonist, significantly decreased the number of calcium peaks per minute in GnRH-1 neurons and was prevented by a Y1R antagonist. Pertussis toxin blocked the effect of NPY on GnRH-1 neuronal activity, indicating the coupling of Y1R to inhibitory G protein. The NPY-induced inhibition was independent of the adenylate cyclase pathway but mediated by the activation of G protein-coupled inwardly rectifying potassium channels. These results indicate that at an early developmental stage, GnRH-1 neuronal activity can be directly inhibited by NPY via its Y1R.