The goal of the present study is to explore whether Ca2+ and Mg2+-binding properties of isomeric Kv channel-interacting proteins (KChIPs) have different effects on their molecular structure and the binding with Kv channel. 8-Anilinonaphthalene- 1-sulfonate fluorescence measurement showed that KChIP4.1 and KChIP2.2 possessed one and two types of Ca2+-binding sites, respectively, and only one type of Mg2+-binding site was noted in the two KChIP proteins. Removal of EF-hand 4 (EF-4) caused a marked drop in their high affinities for Ca2+, but the binding affinity for Mg2+ remained mostly the same. Unlike KChIP4.1, the intact EF-4 was essential for the Kv channel-binding ability of KChIP2.2 in a metal-free buffer. Nevertheless, the interaction of wild-type KChIPs and EF-4-truncated mutants with Kv channel was enhanced by the addition of Mg2+ and Ca2+. In contrast to KChIP4.1, the thermal stability of KChIP2.2 was decreased by the binding of Mg2+ and Ca2+. These results suggest that the conformational change with metal-bound KChIP4.1 is crucial for its interaction with Kv channel but not for KChIP2.2, and that the Mg2+- and Ca2+-binding properties of KChIP2.2 and KChIP4.1 have different effects on their molecular structure.