Since the beginning of investigations of the Na,K-ATPase, it has been well-known that Mg2+ is an essential cofactor for activation of enzymatic ATP hydrolysis without being transported through the cell membrane. Moreover, experimental evidence has been collected through the years that shows that Mg2+ ions have a regulatory effect on ion transport by interacting with the cytoplasmic side of the ion pump. Our experiments allowed us to reveal the underlying mechanism. Mg2+ is able to bind to a site outside the membrane domain of the protein's α subunit, close to the entrance of the access channel to the ion-binding sites, thus modifying the local concentration of the ions in the electrolyte, of which Na+, K+, and H+ are of physiological interest. The decrease in the concentration of these cations can be explained by electrostatic interaction and estimated by the Debye-Hückel theory. This effect provokes the observed apparent reduction of the binding affinity of the binding sites of the Na,K-ATPase in the presence of various Mg2+ concentrations. The presence of the bound Mg2+, however, does not affect the reaction kinetics of the transport function of the ion pump. Therefore, stopped-flow experiments could be performed to gain the first insight into the Na+ binding kinetics on the cytoplasmic side by Mg2+ concentration jump experiments.