There is growing evidence that eicosapentaenoic acid (EPA) uptake has beneficial effects on various cardiovascular diseases. However, electrophysiological actions of EPA remain poorly documented. To investigate the potential antiarrhythmic effects of EPA, mice were fed a high-fat diet (HFD) or an HFD supplemented with EPA for eight weeks. Electrocardiogram (ECG) recordings in combined with esophageal electrical stimulation revealed that HFD-fed mice exhibited bradycardia, reduced P-wave amplitude, and prolonged P-wave duration. Atrial fibrillation (AF) was induced in 100 % of HFD mice, which was only in 50 % of EPA-supplemented mice with significantly shorter durations. HFD-fed mice showed decreased expression of Cav1.2-mRNA, increased expression of Kv1.5-mRNA, elevated expression of inflammatory cytokines (IL-1β, TNF-α, and IL-10), and larger fibrotic area in atrial tissue, which were all reversed by EPA supplementation. These findings suggest that long-term dietary intake of EPA may help maintain normal atrial function and structure, thereby reducing the risk of AF.