Insulin-like peptide 3 (INSL3) is a major peptide hormone produced in a constitutive manner by mature, differentiated, foetal and adult-type Leydig cells, and secreted into the bloodstream, as well as in the adult migrating from interstitial fluid into the seminiferous compartment. This small, approximately 6kD peptide interacts with a large G-protein-coupled receptor, called RXFP2, chiefly responsible for promoting the first transabdominal phase of testicular descent in the foetus. Reduced expression of either INSL3 or RXFP2 leads to cryptorchidism. In the adult, the INSL3/RXFP2 system may also be involved in modulating spermatogenesis, bone metabolism, and horn growth in ruminants, besides having other possible roles in the brain, eye, or kidney. Measurement of INSL3 has shown that it can act as a potent biomarker to monitor foetal development and its perturbation by environmental endocrine disruptors, as well as male pubertal development in a wide range of species, offering a kind of “biochemical Tanner staging”. Circulating INSL3 attains a maximum in the young adult and thereafter declines gradually through middle and old age, where low peripheral concentration may be indicative of hypogonadism and is significantly associated with reduced bone mineral density, loss of sexual function, diabetes, and symptoms of metabolic syndrome.