Abnormal excitation-contraction coupling is a key pathophysiologic component of heart failure (HF), and at a molecular level reduced expression of the sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a) is a major contributor. Previous studies in small animals have suggested that restoration of SERCA function is beneficial in HF. Despite this promise, the means by which this information might be translated into potential clinical application remains uncertain. Using a recently established cardiac-directed recirculating method of gene delivery, we administered adeno-associated virus 2 (AAV2)/1SERCA2a to sheep with pacing-induced HF. We explored the effects of differing doses of AAV2/1SERCA2a (low 1 × 1010 d.r.p.; medium 1 × 1012 d.r.p. and high 1 × 1013 d.r.p.) in conjunction with an intra-coronary delivery group (2.5 × 1013 d.r.p.). At the end of the study, haemodynamic, echocardiographic, histopathologic and molecular biologic assessments were performed. Cardiac recirculation delivery of AAV2/1SERCA2a elicited a dose-dependent improvement in cardiac performance determined by left ventricular pressure analysis, (+dP/dtmax; low dose -220±70, P>0.05; medium dose 125±53, P<0.05; high dose 287±104, P<0.05) and echocardiographically (fractional shortening: low dose -3±2, P>0.05; medium dose 1±2, P>0.05; high dose 6.5±3.9, P<0.05). In addition to favourable haemodynamic effects, brain natriuretic peptide expression was reduced consistent with reversal of the HF molecular phenotype. In contrast, direct intra-coronary infusion did not elicit any effect on ventricular function. As such, AAV2/1SERCA2a elicits favourable functional and molecular actions when delivered in a mechanically targeted manner in an experimental model of HF. These observations lay a platform for potential clinical translation.