Although granulocyte colony-stimulating factor (G-CSF) reportedly plays a cardioprotective role in several models of cardiac injury, clinical use of this drug for cardiac patients has been controversial. Here, we tested the effect of G-CSF in vivo and in vitro on cardiac mitochondria, which play a key role in determining cardiac cellular fate and function. Mild stimulation of C57/BL6 mice with doxorubicin (dox)did not induce cardiac apoptosis or fibrosis, but did induce electron microscopic damages in the mitochondrial alignment of the myocardium. Cardiac catheterization or echocardiogram revealed that dox did not alter cardiac systolic function or left ventricular size, but did reduce diastolic function, an early sign of cardiac damage. Treatment with G-CSF significantly rescued both cardiac mitochondrial changes and diastolic function. In an in vitro model with rat neonatal cardiomyocytes, a sub-apoptotic dose of dox induced severe mitochondrial damage, including markedly swelling of the cardiac mitochondria and/or decreased mitochondrial membrane potential. These mitochondrial changes were completely avoided by pretreatment with G-CSF. In addition, G-CSF dramatically improved ATP generation, rescuing dox-impaired mitochondrial electron transport and oxygen consumption, which was mainly through Complex IV. These findings clearly indicate that G-CSF protects cardiac mitochondria, key organelles in determining cardiac cellular fate, in the early phase of cardiac injury.