Cytochrome P450 (CYP) represents a large family of enzymes that catalyze the oxidation of endogenous and exogenous compounds. The functions of CYP enzymes in the metabolism of xenobiotics have well been established in the liver. However, some CYP enzymes are highly expressed in the heart and catalyze arachidonic acid oxidation to a variety of eicosanoids, which attenuates ischemia-reperfusion injury of the heart. CYP-mediated cardioprotection is associated with activation of multiple pathways such as sarcolemmal and mitochondrial potassium channels, p42/p44 MAPK and PI3K-AKT signaling in cells. CYP enzymes also represent a significant source of reactive oxygen species (ROS) that may target cellular homeostatic mechanisms and mitochondria. CYP isoforms expressed in the heart are critical for generation of epoxyeicosatrienoic acids (EETs) and ROS. It has been demonstrated that CYP2J2 generates cardioprotective EETs, whereas another isozyme in the heart, CYP2C, generates EETs as well as detrimental ROS. Genetic polymorphisms of CYP2C or CYP2J2 have a pathologic impact on coronary artery diseases. Cardiac CYP enzymes can be involved in drug metabolism within the heart and influence pharmacologic efficacy. Metabolism mediated by CYP enzymes influences the survival of cardiomyocytes during ischemia, which is critical for treatment of human ischemic heart disease. In this review, we summarize current knowledge of this enzyme family and discuss the roles of CYP in ischemia-reperfusion injury of the heart.