Cardiac hypertrophy is a major risk factor for congestive heart failure, a leading cause of morbidity and mortality. Abrogating hypertrophic progression is a well-recognized therapeutic goal. Mitochondrial dysfunction is a hallmark of numerous human diseases, including cardiac hypertrophy and heart failure. F1Fo-ATP synthase catalyzes the final step of oxidative energy production in mitochondria. Oligomycin sensitivity conferring protein (OSCP), a key component of the F1Fo-ATP synthase, plays an essential role in mitochondrial energy metabolism. However, the effects of OSCP-targeted therapy on cardiac hypertrophy remain unknown. In the present study, we found that impaired cardiac expression of OSCP is concomitant with mitochondrial dysfunction in the hypertrophied heart. We used cardiac-specific, adeno-associated virus-mediated gene therapy of OSCP to treat mice subjected to pressure overload induced by transverse aortic constriction (TAC). OSCP gene therapy protected the TAC-mice from cardiac dysfunction, cardiomyocyte hypertrophy, and fibrosis. OSCP gene therapy also enhanced mitochondrial respiration capacities in TAC-mice. Consistently, OSCP gene therapy attenuated reactive oxygen species and opening of mitochondrial permeability transition pore in the hypertrophied heart. Together, adeno-associated virus type 9-mediated, cardiac-specific OSCP overexpression can protect the heart via improving mitochondrial function. This result may provide insights into a novel therapy for cardiac hypertrophy and heart failure.