Ablation or inhibition of phospholamban (PLN) has favorable effects in several genetic murine dilated cardiomyopathies, and we showed previously that a pseudophosphorylated form of PLN mutant (S16EPLN) successfully prevented progressive heart failure in cardiomyopathic hamsters. In this study, the effects of PLN inhibition were examined in rats with heart failure after myocardial infarction (MI), a model of acquired disease. S16EPLN was delivered into failing hearts 5 weeks after MI by transcoronary gene transfer using a recombinant adeno-associated virus (rAAV) vector. In treated (MI-S16EPLN, n = 16) and control (MI-saline, n = 18) groups, infarct sizes were closely matched and the left ventricle was similarly depressed and dilated before gene transfer. At 2 and 6 months after gene transfer, MI-S16EPLN rats showed an increase in left ventricular (LV) ejection fraction and a much smaller rise in LV end-diastolic volume, compared with progressive deterioration of LV size and function in MI-saline rats. Hemodynamic measurements at 6 months showed lower LV end-diastolic pressures, with enhanced LV function (contractility and relaxation), lowered LV mass and myocyte size, and less fibrosis in MI-S16EPLN rats. Thus, PLN inhibition by in vivo rAAV gene transfer is an effective strategy for the chronic treatment of an acquired form of established heart failure.