The sodium-potassium (Na/K) pump is a P-type ATPase that generates Na+ and K+ concentration gradients across the cell membrane. For each hydrolyzed ATP molecule, the pump extrudes three Na+ and imports two K+ by alternating between outward- and inward-facing conformations that preferentially bind K+ or Na+, respectively. Remarkably, the selective K+ and Na+ binding sites share several residues, and how the pump is able to achieve the selectivity required for the functional cycle is unclear. Here, free energy–perturbation molecular dynamics (FEP/MD) simulations based on the crystal structures of the Na/K pump in a K+-loaded state (E2·Pi) reveal that protonation of the high-field acidic side chains involved in the binding sites is crucial to achieving the proper K+ selectivity. This prediction is tested with electrophysiological experiments showing that the selectivity of the E2P state for K+ over Na+ is affected by extracellular pH.