The creatine kinase (CK) system is involved in the rapid transport of high-energy phosphates from the mitochondria to the sites of maximal energy requirements such as myofibrils and sarcolemmal ion pumps. Hearts of mice with a combined knockout of cytosolic M-CK and mitochondrial CK (M/Mito-CK^-/-) show unchanged basal left ventricular (LV) performance, but reduced myocardial high-energy phosphate concentrations. Moreover, skeletal muscle from M/Mito-CK^-/- demonstrated altered calcium homeostasis. Our hypothesis was that in CK deficient hearts a cardiac phenotype can be unmasked during acute stress conditions, and that susceptibility to ischemia/reperfusion injury is increased due to altered calcium homeostasis. We simultaneously studied LV performance and myocardial calcium metabolism in isolated perfused hearts of M/Mito-CK^-/- (n=6) and wildtype (WT, n=8) mice during baseline, 20 minutes of no-flow ischemia and recovery. Whereas LV performance was not different during baseline conditions, LV contracture during ischemia developed significantly earlier (408±72 vs. 678±54 sec) and to a greater extent (50±2 vs. 36±3 mmHg) in M/Mito-CK^-/-. During reperfusion, recovery of diastolic function was impaired (LV end-diastolic pressure: 22±3 vs. 10±2 mmHg), while recovery of systolic performance was delayed in M/Mito-CK^-/-. In parallel, calcium transients were similar during baseline conditions, however, M/Mito-CK^-/- showed a greater increase in diastolic [Ca²⁺] during ischemia (237±54 vs. 167±25% of basal [Ca²⁺]) compared to WT. In conclusion, CK deficient hearts show an increased susceptibility of LV performance and calcium homeostasis to ischemic injury, associated with a blunted post-ischemic recovery. This demonstrates a key function of an intact CK system for maintenance of calcium homeostasis and LV mechanics under metabolic stress conditions.