Gene knockout of the KCNJ11-encoded Kir6.2 ATP-sensitive K(+) (K(ATP)) channel implicates this stress-response element in the safeguard of cardiac homeostasis under imposed demand. K(ATP) channels are abundant in ventricular sarcolemma, where subunit expression appears to vary between the sexes. A limitation, however, in establishing the full significance of K(ATP) channels in the intact organism has been the inability to monitor in vivo the contribution of the channel to intracellular calcium handling and the superimposed effect of sex that ultimately defines heart function. Here, in vivo manganese-enhanced cardiac magnetic resonance imaging revealed, under dobutamine stress, a significantly greater accumulation of calcium in both male and female K(ATP) channel knockout (Kir6.2-KO) mice compared with sex- and age-matched wild-type (WT) counterparts, with greatest calcium load in Kir6.2-KO females. This translated, poststress, into a sustained contracture manifested by reduced end-diastolic volumes in K(ATP) channel-deficient mice. In response to ischemia-induced stunning, male and female Kir6.2-KO hearts demonstrated accelerated time to contracture and increased peak contracture compared with WT. The outcome on reperfusion, in both male and female Kir6.2-KO hearts, was a transient reduction in systolic performance, measured as rate-pressure product compared with WT, with protracted increase in left ventricular end-diastolic pressure, exaggerated in female knockout hearts, despite comparable leakage of creatine kinase across groups. Kir6.2-KO hearts were rescued from diastolic dysfunction by agents that target alternative pathways of calcium handling. Thus K(ATP) channel deficit confers a greater susceptibility to calcium overload in vivo, accentuated in female hearts, impairing contractile recovery under various conditions of high metabolic demand.