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The relationships between pheromone stimuli and neuropeptides are not well established in vertebrates due to the limited number of unequivocally identified pheromone molecules. The sea lamprey (Petromyzon marinus) is an advantageous vertebrate model to study the effects of pheromone exposure on neuropeptides since many pheromone molecules and neuropeptides have been identified in this species. Sexually mature male sea lamprey release pheromones 7α, 12α, 24-trihydroxy-5α-cholan-3-one 24-sulfate (3 keto-petromyzonol sulfate, 3kPZS) and 7α, 12α-dihydroxy-5α-cholan-3-one-24-oic acid (3-keto allocholic acid, 3kACA) that differentially regulate gonadotropin-releasing hormone (lGnRH) and steroid levels in sexually immature sea lamprey. However, the effects of these pheromones on gonadotropin-inhibitory hormones (GnIHs), hypothalamic neuropeptides that regulate lGnRH release, are still elusive. In this report, we sought to examine the effects of waterborne pheromones on lamprey GnIH-related neuropeptide levels in sexually immature sea lamprey. Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analyses revealed sex differences in GnIH-related neuropeptide levels in the brain and plasma of immature sea lamprey. Exposure to 3kPZS and 3kACA exerted differential effects on GnIH-related neuropeptide levels in both sexes, but the effects were more prominent in female brains. We conclude that sea lamprey pheromones regulate GnIH-related neuropeptide levels in a sexually dimorphic manner.