The hypothalamus plays a key role in the regulation of both energy homeostasis and reproduction. Evidence suggests that relaxin-3, a recently discovered member of the insulin superfamily, is an orexigenic hypothalamic neuropeptide. Relaxin-3 is thought to act in the brain via the RXFP3 receptor, though the RXFP1 receptor may also play a role. Relaxin-3, RXFP3 and RXFP1 are present in the hypothalamic paraventricular nucleus (PVN), an area with a well characterised role in the regulation of energy balance that also modulates reproductive function by providing inputs to hypothalamic GnRH neurons. Other members of the relaxin family are known to play a role in the regulation of reproduction. However, the effects of relaxin-3 on reproductive function are unknown. We studied the role of relaxin-3 in the regulation of the hypothalamo-pituitary-gonadal (HPG) axis. Intracerebroventricular (ICV, 5 nmol) and intraparaventricular (iPVN, 540-1620 pmol) administration of human relaxin-3 (H3) in adult male Wistar rats significantly increased plasma luteinising hormone (LH) 30 minutes post-injection. This effect was blocked by pre-treatment with a peripheral gonadotropin releasing hormone (GnRH) antagonist. Central administration of human relaxin-2 (H2) showed no significant effect on plasma LH. H3 dose-dependently stimulated the release of GnRH from hypothalamic explants and GT₁-7 cells, which express RXFP1 and RXFP3, but did not influence LH or FSH release from pituitary fragments in vitro. We have demonstrated a novel role for relaxin-3 in the stimulation of the HPG axis, putatively via hypothalamic GnRH neurons. Relaxin-3 may act as a central signal linking nutritional status and reproductive function.