The calcium pump SERCA2a (sarcoplasmic reticulum calcium ATPase 2a), which plays a central role in cardiac contraction, shows decreased expression in heart failure (HF). Increasing SERCA2a expression in HF models improves cardiac function. We used direct cardiac delivery of adeno-associated virus encoding human SERCA2a (AAV6-hSERCA2a) in HF and normal canine models to study safety, efficacy, and the effects of immunosuppression. Tachycardic-paced dogs received left ventricle (LV) wall injection of AAV6-hSERCA2a or solvent. Pacing continued postinjection for 2 or 6 weeks, until euthanasia. Tissue/serum samples were analyzed for hSERCA2a expression (Western blot) and immune responses (histology and AAV6-neutralizing antibodies). Nonpaced dogs received AAV6-hSERCA2a and were analyzed at 12 weeks; a parallel cohort received AAV-hSERCA2a and immunosuppression. AAV-mediated cardiac expression of hSERCA2a peaked at 2 weeks and then declined (to ~50%; p<0.03, 6 vs. 2 weeks). LV end diastolic and end systolic diameters decreased in 6-week dogs treated with AAV6-hSERCA2a (p<0.05) whereas LV diameters increased in control dogs. Dogs receiving AAV6-hSERCA2a developed neutralizing antibodies (titer ≥1:120) and cardiac cellular infiltration. Immunosuppression dramatically reduced immune responses (reduced inflammation and neutralizing antibody titers <1:20), and maintained hSERCA2a expression. Thus cardiac injection of AAV6-hSERCA2a promotes local hSERCA2a expression and improves cardiac function. However, the hSERCA2a protein level is reduced by host immune responses. Immunosuppression alleviates immune responses and sustains transgene expression, and may be an important adjuvant for clinical gene therapy trials.