The sarco(endo)plasmic reticulum calcium ATPase 2 (SERCA2) transports Ca²⁺ from cytosol into the sarcoplasmic reticulum (SR) of cardiomyocytes, thereby maintaining the store of releasable Ca²⁺ necessary for contraction. Reduced SERCA function has been linked to heart failure, and loss of SERCA2 in the adult mammalian heart would be expected to cause immediate severe myocardial contractile dysfunction and death. We investigated heart function in adult mice with an inducible cardiomyocyte-specific excision of the Atp2a2 (Serca2) gene (SERCA2 KO). Seven weeks after induction of Serca2 gene excision, the mice displayed a substantial reduction in diastolic function with a 5-fold increase in the time constant of isovolumetric pressure decay (tau). However, already at 4 weeks following gene excision less than 5% SERCA2 protein was found in myocardial tissue. Surprisingly, heart function was only moderately impaired at this time point. Tissue Doppler imaging showed slightly reduced peak systolic tissue velocity and a less than 2-fold increase in tau was observed. The SR Ca²⁺ content was dramatically reduced in cardiomyocytes from 4-week SERCA2 KO mice, and Ca²⁺ transients were predominantly generated by enhanced Ca²⁺ flux through L-type Ca²⁺ channels and the Na⁺–Ca²⁺ exchanger. Moreover, equivalent increases in cytosolic [Ca²⁺] in control and SERCA2 KO myocytes induced greater cell shortening in SERCA2 KO, suggesting enhanced myofilament responsiveness. Our data demonstrate that SR-independent Ca²⁺ transport mechanisms temporarily can prevent major cardiac dysfunction despite a major reduction of SERCA2 in cardiomyocytes.