Background: Obesity and diets high in saturated fat increase the risk of arrhythmias and sudden cardiac death. However, the molecular mechanisms are not well understood. We hypothesized that an increase in dietary saturated fat could lead to abnormalities of calcium homeostasis and heart rhythm by a NOX2 (NADPH oxidase 2)-dependent mechanism. Methods: We investigated this hypothesis by feeding mice high-fat diets. In vivo heart rhythm telemetry, optical mapping, and isolated cardiac myocyte imaging were used to quantify arrhythmias, repolarization, calcium transients, and intracellular calcium sparks. Results: We found that saturated fat activates NOX (NADPH oxidase), whereas polyunsaturated fat does not. The high saturated fat diet increased repolarization heterogeneity and ventricular tachycardia inducibility in perfused hearts. Pharmacological inhibition or genetic deletion of NOX2 prevented arrhythmogenic abnormalities in vivo during high statured fat diet and resulted in less inducible ventricular tachycardia. High saturated fat diet activates CaMK (Ca 2+ /calmodulin-dependent protein kinase) in the heart, which contributes to abnormal calcium handling, promoting arrhythmia. Conclusions: We conclude that NOX2 deletion or pharmacological inhibition prevents the arrhythmogenic effects of a high saturated fat diet, in part mediated by activation of CaMK. This work reveals a molecular mechanism linking cardiac metabolism to arrhythmia and suggests that NOX2 inhibitors could be a novel therapy for heart rhythm abnormalities caused by cardiac lipid overload.