Doxorubicin (DXR) is a widely used and efficient anticancer drug. However, its application is limited by the risk of severe cardiotoxicity. Impairment of cardiac high-energy phosphate homeostasis is an important manifestation of both, acute and chronic DXR cardiotoxic action. Using the Langendorff model of perfused rat heart, we have characterized acute effects of 1 h perfusion with 2 or 20 µM DXR on two key-kinases in cardiac energy metabolism, creatine kinase (CK) and AMP-activated protein kinase (AMPK), and related them to functional responses of the perfused heart, structural integrity of the contractile apparatus, as well as drug accumulation in cardiomyocytes. DXR-induced changes in CK were dependent on the isoenzyme, with a shift in protein levels of cytosolic isoenzymes from MCK to BCK, and a destabilization of octamers of the mitochondrial isoenzyme (sMtCK) accompanied by drug accumulation in mitochondria. Interestingly, DXR rapidly reduced protein level and phosphorylation of AMPK, as well as phosphorylation of its target acetyl-CoA-carboxylase. AMPK was strongly affected already at 2 µM DXR, even before substantial cardiac dysfunction occurred. Impairment of CK isoenzymes was mostly moderate, but became significant at 20 µM DXR. Only at 2 µM DXR, upregulation of BCK compensated for inactivation of other isoenzymes. These results suggest that an impairment of kinase systems regulating cellular energy homeostasis is involved in the development of DXR cardiotoxicity.