Insulin-like peptide 3 (INSL3) is a biomarker for Leydig cells in the testes of vertebrates, and it is principally involved in spermatogenesis through specific binding with the RXFP2 receptor. This study reports the gene transcript and the Insl3 prepropeptide expression in both non-reproductive and reproductive tissues of . An immunohistochemistry analysis shows that the hormone is present at a low level in the Leydig cells and germ cells at all stages of testis differentiation. Considering that the gene is transcribed in Leydig cells, our results highlight an autocrine and paracrine function of this hormone in the testis, adding new information on the Insl3 mode of action in reproduction. We also show that Insl3 and Rxfp2 belonging to and other vertebrate species share most of the amino acid residues involved in the ligand-receptor interaction and activation, suggesting a conserved mechanism of action during vertebrate evolution.