<jats:sec> <jats:title>Background—</jats:title> <jats:p> Recently, we reported that sarcoplasmic reticulum Ca <jats:sup>2+</jats:sup> ATPase 2a (SERCA2a), the pump responsible for reuptake of cytosolic calcium during diastole, plays a central role in the molecular mechanism of cardiac alternans. Heart failure (HF) is associated with impaired myocardial calcium handling, deficient SERCA2a, and increased susceptibility to cardiac alternans. Therefore, we hypothesized that restoring deficient SERCA2a by gene transfer will significantly reduce arrhythmogenic cardiac alternans in the failing heart. </jats:p> </jats:sec> <jats:sec> <jats:title>Methods and Results—</jats:title> <jats:p> Adult guinea pigs were divided into 3 groups: control, HF, and HF+AAV9.SERCA2a gene transfer. HF resulted in a decrease in left ventricular fractional shortening compared with controls ( <jats:italic>P</jats:italic> <0.001). As expected, isolated HF myocytes demonstrated slower sarcoplasmic reticulum calcium uptake, decreased Ca <jats:sup>2+</jats:sup> release, and increased diastolic Ca <jats:sup>2+</jats:sup> ( <jats:italic>P</jats:italic> <0.05) compared with controls. Moreover, SERCA2a, cardiac ryanodine receptor 2, and sodium-calcium exchanger protein expression was decreased in HF compared with control ( <jats:italic>P</jats:italic> <0.05). As predicted, HF increased susceptibility to cardiac alternans, as evidenced by decreased heart rate thresholds for both V <jats:sub>m</jats:sub> alternans and Ca alternans compared with controls ( <jats:italic>P</jats:italic> <0.01). Interestingly, in vivo gene transfer of AAV9.SERCA2a in the failing heart improved left ventricular contractile function ( <jats:italic>P</jats:italic> <0.01), suppressed cardiac alternans ( <jats:italic>P</jats:italic> <0.01), and reduced ryanodine receptor 2 P <jats:sub>o</jats:sub> secondary to reduction of ryanodine receptor 2–P <jats:sub>S2814</jats:sub> ( <jats:italic>P</jats:italic> <0.01). This ultimately resulted in a decreased incidence of inducible ventricular arrhythmias ( <jats:italic>P=</jats:italic> 0.05). </jats:p> </jats:sec> <jats:sec> <jats:title>Conclusions—</jats:title> <jats:p> These data show that SERCA2a gene transfer in the failing heart not only improves contractile function but also directly restores electric stability through the amelioration of key arrhythmogenic substrate (ie, cardiac alternans) and triggers (ie, sarcoplasmic reticulum Ca <jats:sup>2+</jats:sup> leak). </jats:p> </jats:sec>