Several cytochrome P450 (P450) enzymes have been identified in the heart, and their levels have been reported to be altered during cardiac hypertrophy. Moreover, there is a strong correlation between P450-mediated arachidonic acid metabolites and the pathogenesis of cardiac hypertrophy. Therefore, we investigated the effect of isoproterenol-induced cardiac hypertrophy on the expression of several P450 genes and their associated P450-derived metabolites of arachidonic acid. Cardiac hypertrophy was induced by seven daily i.p. injections of 5 mg/kg isoproterenol. Thereafter, the heart, lung, liver, and kidney were harvested, and the expression of different genes was determined by real-time polymerase chain reaction. Heart microsomal protein from control or isoproterenol treated rats was incubated with 50 μM arachidonic acid, and arachidonic acid metabolites were determined by liquid chromatography-electron spray ionization-mass spectrometry. Our results show that isoproterenol treatment significantly increased the heart/body weight ratio and the hypertrophic markers. In addition, there was a significant induction of CYP1A1, CYP1B1, CYP4A3, and soluble epoxide hydrolase and a significant inhibition of CYP2C11 and CYP2E1 in the hypertrophied hearts as compared with the control. CYP1A1, CYP2E1, and CYP4A3 gene expression was induced in the kidney, and CYP4A3 was induced in the liver of isoproterenol-treated rats. Isoproterenol treatment significantly reduced 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid formation and significantly increased their corresponding 8,9-, and 14,15-dihydroxyeicosatrienoic acid and the 20-hydroxyeicosatetraenoic acid metabolite. In conclusion, isoproterenol-induced cardiac hypertrophy alters arachidonic acid metabolism and its associated P450 enzymes, suggesting their role in the development and/or progression of cardiac hypertrophy.