Calumenin (Calu) is a well-conserved multi-EF-hand-containing Ca2+-binding protein. In this work, we focused on the alterations that calumenin has undergone during evolution. We demonstrate that vertebrate calumenin is significantly different from its invertebrate homologues with respect to its response to Ca2+ binding. Human calumenin (HsCalu1) is intrinsically unstructured in the Ca2+ free form and responds to Ca2+ with a dramatic gain in structure. Calumenin from Caenorhabditis elegans (CeCalu) is structured even in the apo form, with no conformational change upon binding of Ca2+. We decode this structural and functional distinction by identifying a single "Leu" residue-based switch located in the fourth EF-hand of HsCalu1, occupied by "Gly" in the invertebrate homologues. We demonstrate that replacing Leu with Gly (L150G) in HsCalu1 enables the protein to adopt a structural fold even in the Ca2+ free form, similar to CeCalu, leading to ligand compensation (adoption of structure in the absence of Ca2+). The fourth (of seven) EF-hand of HsCalu1 nucleates the structural fold of the protein depending on the switch residue (Gly or Leu). Our analyses reveal that the Leu that replaced Gly from fishes onward is absolutely conserved in higher vertebrates, while lower organisms have Gly, not only enlarging the scope of Ca2+-dependent structural transitions but also drawing a boundary between the invertebrate and vertebrate calumenin. The evolutionary selection of the switch residue strongly corroborates the change in the structure of the protein and its pleiotropic functions and seems like it can be extended to the presence or absence of a heart in that organism.