Folding and structural stability are key factors for the proper biological function of proteins. Na+,K+-ATPase is an integral membrane protein involved in the active transport of Na+ and K+ across the plasma membrane. In this work we characterized the effects of K+ and Na+ on the thermal inactivation of Na+,K+-ATPase, evaluating both catalytic and transport capacities of the pump. Both activities of the enzyme decrease with the preincubation time as first-order kinetics. The thermal inactivation of Na+,K+-ATPase is simultaneous with a conformational change detected by tryptophan and 1-aniline-8-naphtalenesulfonate (ANS) fluorescence. The kinetic coefficient of thermal inactivation was affected by the presence of Na+ and K+ (or Rb+) and the temperature of the preincuabtion media. Our results show that K+ or Rb+ stabilize the enzyme, while Na+ decreases the stability of Na+,K+-ATPase. Both effects are exerted by the specific binding of these cations to the pump. Also, we provided strong evidence that the Rb+ (or K+) stabilization effect is due to the occlusion of these cations into the enzyme. Here, we proposed a minimal kinetic model that explains the behavior observed in the experimental results and allows a better understanding of the results presented by other researchers. The thermal inactivation process was also analyzed according to Kramer's theory.