Objective: Endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) have been implicated in protection against myocardial ischemia injury. This study was designed to explore a new method of therapy for myocardial injury by eNOS gene transfection. Methods: A rat model of myocardial infarction (MI) was established by left anterior descending (LAD) coronary artery ligation. eNOS gene in an adenovirus vector was delivered locally into the rat heart and hemodynamic parameters were examined after 3 weeks, Matrix metalloproteinase-2 and 9 (MMP-2, MMP-9) mRNA were measured by reverse transcription polymerase chain reaction (RT-PCR), and the protein levels of eNOS, caspase-3, and transforming grouth factor β1 (TGF-β1) were determined by western blot assay. Results: eNOS gene transfer significantly reduced cardiomyocyte apoptosis and improved cardiac function. In addition, eNOS significantly reduced the mRNA levels of MMP-2 and MMP-9. In the eNOS gene transfected group, the activation of caspase-3 and TGF-β1 were decreased. However, the protection was reversed by administration of the NOS inhibitor, N(ω)-nitro-l-arginine methyl ester (L-NAME). Conclusion: These results demonstrate that the eNOS provides cardiac protection after myocardial infarction injury through inhibition of cardiac apoptosis and collagen deposition, and suppression of TGF-β1.