Cardiac-restricted overexpression of the Ca2+-binding protein S100A1 has been shown to lead to increased myocardial contractile performance in vitro and in vivo. Since decreased cardiac expression of S100A1 is a characteristic of heart failure, we tested the hypothesis that S100A1 gene transfer could restore contractile function of failing myocardium. Adenoviral S100A1 gene delivery normalized S100A1 protein expression in a postinfarction rat heart failure model and reversed contractile dysfunction of failing myocardium in vivo and in vitro. S100A1 gene transfer to failing cardiomyocytes restored diminished intracellular Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load mechanistically due to increased SR Ca2+ uptake and reduced SR Ca2+ leak. Moreover, S100A1 gene transfer decreased elevated intracellular Na+ concentrations to levels detected in nonfailing cardiomyocytes, reversed reactivated fetal gene expression, and restored energy supply in failing cardiomyocytes. Intracoronary adenovirus-mediated S100A1 gene delivery in vivo to the postinfarcted failing rat heart normalized myocardial contractile function and Ca2+ handling, which provided support in a physiological context for results found in myocytes. Thus, the present study demonstrates that restoration of S100A1 protein levels in failing myocardium by gene transfer may be a novel therapeutic strategy for the treatment of heart failure.