Cardiac endothelial cells (ECs) are important targets for cardiovascular gene therapy. However, the approach of stably transducing ECs using different vectors, including adeno-associated virus (AAV), remains unexamined. Regarding this unmet need, two AAV libraries from DNA shuffling and random peptide display were simultaneously screened in a transgenic mouse model. Cardiac ECs were isolated by cell sorting for salvage of EC-targeting AAV. Two AAV variants, i.e., EC71 and EC73, enriched in cardiac EC, were further characterized for their tissue tropism. Both of them demonstrated remarkably enhanced transduction of cardiac ECs and reduced infection of liver ECs in comparison to natural AAVs after intravenous injection. Significantly, persistent transgene expression was maintained in mouse cardiac ECs for at least 4?months. The EC71 vector was selected for delivery of the endothelial nitric oxide synthase (eNOS) gene into cardiac ECs in a mouse model of myocardial infarction. Enhanced eNOS activity was observed in the mouse heart and lung, which was correlated with partially improved cardiac function. Taken together, two AAV capsids were evolved with more efficient transduction in cardiovascular endothelium , but their endothelial tropism might need to be further optimized for practical application to cardiac gene therapy.