Objective— The protective effect of NO synthase-3 (eNOS)–derived NO in limiting myocardial ischemia-reperfusion (MI-R) injury is well established. We reported previously that systemic genetic overexpression of eNOS attenuates MI-R injury. The purpose of the current study was to investigate tissue-specific genetic overexpression of the human eNOS gene. Methods and Results— To accomplish this, we used 2 distinct murine models of transgenic overexpression, a cardiomyocyte-specific eNOS overexpresser (CS eNOS-Tg) under the control of the α-myosin heavy chain promoter, and a systemic eNOS transgenic mouse (SYS eNOS-Tg) under control of the native eNOS promoter with an upstream endothelial enhancer element. Mice were subjected to 30 or 45 minutes of left coronary artery ischemia and 24 or 72 hours of reperfusion. CS eNOS-Tg mice displayed significantly decreased infarct size beyond that of mice with systemic overexpression. Additionally, CS eNOS-Tg mice exhibited better preservation of cardiac function compared with SYS eNOS-Tg mice after myocardial infarction. Conclusion— These results provide evidence that site-specific targeting of eNOS gene therapy may be more advantageous in limiting MI-R injury and subsequent cardiac dysfunction.