The only input to total iron in mammalian systems, other than by clinical intervention is through diet. Because there exists no mechanism of controlled iron removal from mammals, iron regulation by physiological and biochemical recycling mechanisms, finely balanced by controlled dietary intake, each mediated by the peptide hormone hepcidin-25 (herein hepcidin) are integral to maintaining the strict requirements of iron homeostasis. With the physiological significance of this hormone well established, it has emerged as an informative biomarker. The use of laboratory mice as models of iron metabolism has greatly advanced our understanding of hepcidin and iron regulatory processes more broadly. It’s recognised that an availability of animal models with a more representative iron turnover to that of humans would also be advantageous for human translation. More directly, hepcidin may yet prove useful for animal diagnostics. Here we reveal the first measurement of hepcidin from dogs by mass spectrometry and determine that previous reports of the amino acid sequence were contradictory to these data. Measurement of the peptide by mass spectrometry, following isolation from greyhound blood serum, revealed an amino acid sequence and peptide mass, differing from all accounts to date, yet demonstrating perfect sequence identity to that of the greater Canidae lineage of the Carnivora, as predicted by genome annotation. In the greyhound, the measured hepcidin peptide showed a similar temporal pattern to total serum iron, consistent with our understanding of hepcidin regulation of iron homeostasis, in agreement with expectations of human diagnostics, and providing translational evidence of the measured peptide being the iron regulatory hormone of the Canidae.