Background: Supplementing animal diets with fish oil increases myocardial omega-3 polyunsaturated fatty acids [ω-3 (n-3) PUFA], lowers heart rate, and prevents malignant cardiac arrhythmias. In contrast to epidemiological reports, results of some human clinical trials and of unphysiologically high doses employed in animal studies call into question the application of dietary ω-3 PUFA for cardioprotection. Objective: This study tested the hypothesis that low ω-3 PUFA dietary thresholds for myocardial incorporation in rats, equivalent in dose to what humans derive from eating fish, can reduce heart rate and arrhythmia vulnerability. Methods: Male Sprague-Dawley rats (12-15 wk old) were fed isoenergetic diets containing 10% fat for 4-5 wk. The control diet (CON) contained 5.5% beef tallow, 2.5% sunflower seed oil, and 2% olive oil. Fish oil diets contained high-DHA tuna oil, exchanged for olive oil: 0.31% [fish oil group 1 (FO1)] (human equivalent EPA + DHA 570 mg/d); 1.25% [fish oil group 2 (FO2)] (equivalent EPA + DHA 2.3 g/d). Anaesthetized rats (pentobarbital, 60 mg/kg intraperitoneally) were subjected in vivo to 15-min cardiac ischemia by left coronary artery occlusion and then reperfusion, with arrhythmias detected by electrocardiogram. Results: Fish oil dose dependently modulated myocardial membrane fatty acids (DHA mean ± SEM: CON, 5.0 ± 0.2%; FO1, 13.1 ± 0.9%; FO2, 18.3 ± 0.4%; n = 4-5; P-trend < 0.001 ANOVA); resting heart rate (CON, 453 ± 6; FO1, 432 ± 4; FO2, 422 ± 5 bpm; n = 15-18; P-trend < 0.001); reduced ventricular fibrillation (VF) (CON, 89%; FO1, 60%; P = 0.052; FO2, 50%; n = 15-18; P = 0.013 chi square); and total arrhythmia severity (arrhythmia score: CON, 6.1 ± 0.4; FO1, 4.6 ± 0.5; FO2, 3.1 ± 0.7; n = 15-18; P-trend < 0.01) during ischemia and reperfusion (VF: Con, 86%; FO1, 22% P = 0.011; FO2, 8% P = 0.001; n = 7-12); (arrhythmia score: CON, 4.6 ± 0.3; FO1, 3.1 ± 0.3; FO2, 1.3 ± 0.3; n = 7-12; P-trend < 0.001). Conclusions: Ventricular arrhythmias were prevented and heart rate was slowed by lower ω-3 PUFA intake in rats than previously reported, equivalent to human fish consumption and associated with increased myocardial DHA. The efficacy of low-dose fish oil demonstrates biological plausibility for nutritional ω-3 fatty acid-mediated cardioprotection and suggests that effectiveness in human clinical trials may be obscured by failure to exclude fish eaters.