Abstract: The magnetic sense, or termed as magnetoreception, has been evolved in a broad range of taxa in animal kingdom for orientation and navigation. MagR, a highly conserved A-type iron-sulfur protein widely distributed across all phyla, plays essential roles in both magnetoreception and iron-sulfur cluster biogenesis. How MagR evolved from its prokaryotic ancestor and confer diverse functions remains unclear. Here in this study, MagR sequences from 131 species from bacteria to human were selected for analysis, and 23 representative sequences covering species from prokaryotes to mollusca, arthropoda, osteichthyes, reptilia, aves and mammalians, were chosen for protein expression and purification. Further biochemical studies demonstrated the total iron content gradually increased in MagR during evolution, and three types of MagRs were then identified with distinctive iron, or iron and iron-sulfur cluster binding capacity and protein stability, which indicate MagR’s functional roles have been continuously enhanced and expanded during speciation and evolution. The evolutionary biochemical studies presented here allow us to piece together how evolution shapes the physical and chemical properties of a biological molecule such as MagR and how those properties shape evolutionary trajectories of MagR as well.