Interventions involving calcium cycling may represent a promising approach to heart failure (HF) therapy because calcium handling is known to be deranged in human and experimental HF. Istaroxime is a sodium-potassium adenosine triphosphatase (ATPase) inhibitor with the unique property of increasing sarcoplasmic reticulum calcium ATPase (SERCA) isoform 2a (SERCA2a) activity. Because this was demonstrated in normal experimental models, we investigated whether istaroxime is able to improve global cardiac function and stimulate SERCA in failing hearts. In guinea pigs with 3-month aortic banding (AoB), echocardiographic results showed that istaroxime intravenous infusion (0.11 mg/kg per min) significantly increased both indices of contraction and relaxation (fractional shortening, +18 ± 3.7%; aortic flow rate, +19 ± 2.9%; peak myocardial systolic velocity, +36 ± 7%; circumferential fiber shortening, +24 ± 4.1%; peak atrial flow velocity, +69 ± 8.6%; isovolumic relaxation time, +19 ± 6.9%; and peak myocardial early diastolic velocity, +42 ± 12%). In left ventricular sarcoplasmic reticulum microsomes from AoB animals, 100 nmol/L istaroxime normalized the depressed (−32%) SERCA2a maximum velocity and increased SERCA activity (+17%). In muscle strips from hearts from patients undergoing cardiac transplantation, istaroxime (0.1–1.0 μmol/L) increased (in a concentration-dependent manner) developed tension, the maximum and minimum first derivative of tension, and absolute velocity of contraction, while stimulating SERCA activity in sarcoplasmic reticulum microsomes at physiologic free calcium concentrations. In conclusion, istaroxime is presently the only available compound that stimulates SERCA2a activity and produces a luso-inotropic effect in HF.