Na+,K+-ATPase is the ion pump responsible for maintenance of the electrochemical gradients of Na+ and K+ across the membrane of animal cells. Cardiotonic steroids constitute a broad class of specific Na+,K+-ATPase inhibitors, including drugs of clinical importance with multiple physiological effects. The existence of several endogenous cardiotonic steroids suggests their involvement in health and disease mediated by various signaling pathways, but the structure–activity relationships are not yet understood. In this study, using X-ray crystallography and analysis of binding kinetics, we characterize Na+,K+-ATPase complexes with a total of five cardiotonic steroids, showing variations in glycosylation, steroid core substituents, and structure of the lactone substituent. The finding reveals that structural features of the pump are altered by the binding of cardiac glycosides. The alteration depends on the steroid’s structural details as well as the cations present in the pump. This insight is highly relevant for the understanding of physiological effects and future drug development based on cardiotonic steroids.