Background: Wnt signaling plays a critical role in both embryonic cardiogenesis and cardiac remodeling in adult heart disease. We have previously demonstrated that the canonical Wnt/β-catenin pathway inhibits cardiac sodium current, but it remains unclear whether the noncanonical Wnt pathway affects cardiac electrophysiology. Methods and Results: Western blot analysis of ventricular tissues from patients with heart failure (n=6) demonstrated a 2.3x fold increase (p<0.01) in the protein level of Wnt5a, a noncanonical Wnt ligand, as compared to healthy ventricular tissues (n=5). To investigate if Wnt5a affects cardiac electrophysiology, adenovirus expressing Wnt5a and mCherry (Ad-Wnt5a) or control adenovirus expressing mCherry only (Ad-mCherry) was injected into the left ventricular free wall of adult rat hearts. At 4-5 days after virus injection, surface ECG revealed increased QT interval (p<0.01) in Ad-Wn5a-injected rats (90.1±2.3 ms n=7, vs 72.3±2.0 ms in control Ad-mCherry rats n=7). In addition, ventricular tachycardia was induced by programmed electrical stimulation in 92% (11/12) Ad-Wnt5a hearts, but only in 22% (2/9) control Ad-mCherry hearts (p<0.01). Patch-clamp recording of isolated single ventricular myocytes demonstrated that Ad-Wnt5a myocytes exhibited marked prolongation of action potential duration (APD 90 : 273±77ms, n=5) as compared to control cells (42±12 ms, n=7, p<0.05). In addition, the prolonged action potentials in Ad-Wnt5a myocytes were associated with frequent early afterdepolarizations and delayed afterdepolarizations, two mechanisms for triggered ventricular arrhythmias. Conclusion: Wnt5a is increased in the myocardium of patients with heart failure. Viral expression of Wnt5a in rat ventricular tissue increases QT interval and ventricular arrhythmia susceptibility, which is associated with prolongation of action potentials in cardiomyocytes. This may be an important target for future therapies.