Cyclophilin D (which is encoded by the Ppif gene) is a mitochondrial matrix peptidyl-prolyl isomerase known to modulate opening of the mitochondrial permeability transition pore (MPTP). Apart from regulating necrotic cell death, the physiologic function of the MPTP is largely unknown. Here we have shown that Ppif^–/– mice exhibit substantially greater cardiac hypertrophy, fibrosis, and reduction in myocardial function in response to pressure overload stimulation than control mice. In addition, Ppif^–/– mice showed greater hypertrophy and lung edema as well as reduced survival in response to sustained exercise stimulation. Cardiomyocyte-specific transgene expression of cyclophilin D in Ppif^–/– mice rescued the enhanced hypertrophy, reduction in cardiac function, and rapid onset of heart failure following pressure overload stimulation. Mechanistically, the maladaptive phenotype in the hearts of Ppif^–/– mice was associated with an alteration in MPTP-mediated Ca²⁺ efflux resulting in elevated levels of mitochondrial matrix Ca²⁺ and enhanced activation of Ca²⁺-dependent dehydrogenases. Elevated matrix Ca²⁺ led to increased glucose oxidation relative to fatty acids, thereby limiting the metabolic flexibility of the heart that is critically involved in compensation during stress. These findings suggest that the MPTP maintains homeostatic mitochondrial Ca²⁺ levels to match metabolism with alterations in myocardial workload, thereby suggesting a physiologic function for the MPTP.