Recently, gonadotropin‐inhibitory hormone (GnIH) has emerged as an important regulator of reproduction in birds and mammals. This RFamide neuropeptide has neuromodulatory functions and controls the synthesis and/or release of gonadotropin‐releasing hormone (GnRH) and gonadotropins. Although teleosts represent about half of all living vertebrates, scientific and technological advances on the Gnih system in fish are scarce, contradictory, and inconclusive. Research on the fish Gnih system appears necessary to better clarify its role in the neuroendocrine and environmental control of vertebrate reproduction. In this study, we cloned a full‐length sequence for the Gnih precursor of a flatfish, the Senegalese sole, coding for three putative Gnih peptides (ssGnih). We also generated specific antibodies against these ssGnih peptides, and used them to localize Gnih cells and their projections in the brain and pituitary. The expression of gnih was particularly evident in the diencephalon, but also in the olfactory bulbs/cerebral hemispheres, optic tectum/tegmentum, retina, and pituitary. The three antibodies used provided consistent results and showed that ssGnih‐immunoreactive perikarya were present in the olfactory bulbs, ventral telencephalon, caudal preoptic area, dorsal tegmentum and rostral rhombencephalon, and their fibers innervated the brain and pituitary profusely. Intramuscular injection of ssGnih‐3 provoked a significant reduction in gnrh‐3 and lh expression, whereas ssGnih‐2 treatment did not affect transcript levels of the main reproductive genes. Our results reveal the existence of a functional Gnih system in the sole brain, profusely innervating different brain areas and the pituitary gland, which could represent an important factor in the neuroendocrine control of flatfish reproduction. In the present study, we cloned a Gnih precursor encoding three putative Gnih orthologs in a flatfish species, the Senegalese sole. We also generated specific antibodies against these ssGnih peptides, and used them to localize Gnih cells and their projections in the brain and pituitary. Finally, we have shown that ssGnih‐3 (but not ssGnih‐2) inhibits the expression of a hypophysiotropic Gnrh (gnrh‐3) and the maturational gonadotropin (lh), which provide support to its role in the control of reproduction in sole.