Recent studies have demonstrated that liver-expressed antimicrobial peptide 2 (LEAP2) antagonizes the ghrelin receptor GHSR1a in mammals. However, its antagonistic function in lower vertebrates has not yet been tested. LEAP2 orthologs have been identified from a variety of fish species; however, previous studies all focused on their antimicrobial activity. To test whether LEAP2 functions as a GHSR1a antagonist in the lowest vertebrates, we studied the antagonism of a fish LEAP2 from Latimeria chalumnae, an extant coelacanth that is one of the closest living fish relatives of tetrapods. Using binding assays, we demonstrated that the coelacanth LEAP2 and ghrelin bound to the coelacanth GHSR1a with IC₅₀ values in the nanomolar range. Using activation assays, we demonstrated that the coelacanth ghrelin activated the coelacanth GHSR1a with an EC₅₀ value in the nanomolar range, and this activation effect was efficiently antagonized by a nanomolar range of the coelacanth LEAP2. In addition, we also showed that the human LEAP2 and ghrelin were as effective as their coelacanth orthologs towards the coelacanth GHSR1a; however, the coelacanth peptides had moderately lower activity towards the human GHSR1a. Thus, LEAP2 serves as an endogenous antagonist of the ghrelin receptor GHSR1a in coelacanth and the ghrelin-LEAP2-GHSR1a system has evolved slowly since its emergence in ancient fish.