Nitric oxide modulates the severity of myocardial ischemia–reperfusion (I/R) injury. We investigated whether cardioselective nitric oxide synthase 3 (NOS3) gene transfer could confer myocardial protection against I/R injury in pigs and examined potential molecular mechanisms. I/R injury was induced by balloon occlusion of the left anterior descending artery for 45 min followed by 4 or 72 h reperfusion. Hemodynamic and pathological changes were measured in pigs in the absence (n = 11) or presence of prior intracoronary retroinfusion of human NOS3 (AdNOS3, 5 × 10¹⁰ PFU, n = 13) or control vector (AdRR5, 5 × 10¹⁰ PFU, n = 11). Retrograde NOS3 gene transfer selectively increased NOS3 expression and NO bioavailability in the area at risk (AAR) without changing endogenous NOS isoform expression. At 4 h R, LV systolic (dP/dt _max) and diastolic (dP/dt _min) function was better preserved in AdNOS3- than in AdRR5-injected pigs (2,539 ± 165 vs. 1,829 ± 156 mmHg/s, and −2,781 ± 340 vs. −2,062 ± 292 mmHg/s, respectively, P < 0.05 for both). Myocardial infarct size (% AAR) was significantly smaller in AdNOS3 than in control and AdRR5 and associated with a significantly greater reduction in cardiac myeloperoxidase activity, a marker of neutrophil infiltration. The latter effects were sustained at 72 h R in a subset of pigs (n = 7). In the AAR, intercellular endothelial adhesion molecule-1 expression and cardiomyocyte apoptosis were significantly lower in AdNOS3. In conclusion, single myocardial NOS3 retroinfusion attenuates I/R injury, and causes a sustained reduction in myocardial infarct size and inflammatory cell infiltration. Gene-based strategies to increase NO bioavailability may have therapeutic potential in myocardial I/R.