In 2000, we discovered a novel hypothalamic neuropeptide that actively inhibits gonadotropin release in quail and termed it gonadotropin-inhibitory hormone (GnIH).¹ GnIH peptides have since been identified in most representative species of gnathostomes.² They all share a C-terminal LPXRFamide (X = L or Q) motif.² GnIH can inhibit gonadotropin synthesis and release by decreasing the activity of GnRH neurons as well as by directly inhibiting pituitary gonadotrope activity in birds and mammals.² To investigate the evolutionary origin of GnIH and its ancestral function, we identified GnIH precursor gene encoding GnIHs from the brain of sea lamprey, the most ancient lineage of vertebrates.³ Lamprey GnIHs possessed a C-terminal PQRFa motif.³ One of lamprey GnIHs stimulated the expression of lamprey GnRH in the hypothalamus and GTHβ mRNA expression in the pituitary.³ Thus, GnIH may have emerged in agnathans as a stimulatory neuropeptide that later diverged to an inhibitory neuropeptide during the course of evolution from basal vertebrate to later-evolved vertebrates, such as birds and mammals. From a structural point of view, pain modulatory neuropeptides, such as neuropeptide FF (NPFF) and NPAF, share a C-terminal PQRFa motif. Because we identified GnIH and NPFF genes from the brain of agnathans, the origin of GnIH and NPFF genes may date back before the emergence of early vertebrates.^3,4 Recently, we identified a novel gene encoding RFamide peptides in the amphioxus. Molecular phylogenetic analysis and synteny analysis indicated that this gene is closely related to the genes of GnIH and NPFF of vertebrates. The identified protochordate gene is considered to be a common ancestral form of GnIH and NPFF genes, suggesting that the origin of GnIH and NPFF may date back to the time of the emergence of early chordates. GnIH gene and NPFF gene may have diverged by whole-genome duplication in the course of vertebrate evolution.