BackgroundOmega-3 polyunsaturated fatty acids (ω3-PUFAs) from fish oil (FO) reduce heart rate in humans. The mechanism underlying this cardioprotective effect of FO is unknown.ObjectiveWe studied the effects of an FO diet on heart rate, pacemaker activity, and pacemaker current (If) in sinoatrial node (SAN) cells.MethodsRabbits were fed a diet enriched with 2.5% (w/w) FO or 2.5% high oleic sunflower oil (SO) as control for 3 weeks. Heart rate was measured in Langendorff-perfused hearts. Pacemaker activity and If were recorded using the perforated patch-clamp technique in isolated SAN cells.ResultsIn SAN cells, the FO diet reduced If density by approximately 30%, without changes in its voltage dependence, reversal potential, (de)activation kinetics, and sensitivity to β-stimulation. Dietary FO significantly prolonged the cycle length in both isolated perfused hearts (337 ± 8 [mean ± SEM, n = 8] vs. 301 ± 9 ms [n = 8]) and single cells (363 ± 20 [n = 19] vs. 276 ± 8 ms [n = 22]). In single cells, dietary FO significantly decreased the diastolic depolarization rate by 33% and prolonged the action potential by 27%, whereas other action potential parameters were unaltered. If blockade experiments substantiated that the reduced diastolic depolarization rate in the FO group was partially caused by the If density reduction.ConclusionAn FO diet prolongs the sinus cycle length because of, at least in part, a reduction in If density. Our results explain FO-induced heart rate reduction and suggest FO as an alternative or adjunct to If-lowering drugs.