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All known protein kinases, except CASK [calcium/calmodulin (CaM)–activated serine-threonine kinase], require magnesium ions (Mg2+) to stimulate the transfer of a phosphate from adenosine 5′-triphosphate (ATP) to a protein substrate. The CaMK (calcium/calmodulin-dependent kinase) domain of CASK shows activity in the absence of Mg2+; indeed, it is inhibited by divalent ions including Mg2+. Here, we converted the Mg2+-inhibited wild-type CASK kinase (CASKWT) into a Mg2+-stimulated kinase (CASK4M) by substituting four residues within the ATP-binding pocket. Crystal structures of CASK4M with and without bound nucleotide and Mn2+, together with kinetic analyses, demonstrated that Mg2+ accelerates catalysis of CASK4M by stabilizing the transition state, enhancing the leaving group properties of adenosine 5′-diphosphate, and indirectly shifting the position of the γ-phosphate of ATP. Phylogenetic analysis revealed that the four residues conferring Mg2+-mediated stimulation were substituted from CASK during early animal evolution, converting a primordial, Mg2+-coordinating form of CASK into a Mg2+-inhibited kinase. This emergence of Mg2+ sensitivity (inhibition by Mg2+) conferred regulation of CASK activity by divalent cations, in parallel with the evolution of the animal nervous systems.