Omega-3 polyunsaturated fatty acids in fish oils may have antifibrillatory effects. Their mechanism of action in humans is poorly understood.The purpose of this study was to investigate the effects of chronic fish oil supplementation on human atrial electrophysiology (EP).Two groups of patients without clinical AF or structural heart disease and fish intake ≤1/week were prospectively recruited into a control group (n = 30) and a fish oil group (n = 31). The latter were prescribed 6 g/day of fish oil for ≥1 month before an EP study. The following were compared at time of EP study: serum omega-3 levels; right atrial and coronary sinus effective refractory periods (ERPs); interatrial, intra-atrial, left atrial, and coronary sinus conduction at baseline and the maximal conduction delay with the shortest propagated extrastimulus; and inducibility of AF (10 inductions/patient).The following significant differences were noted favoring the fish oil group at time of EP: (1) twofold higher total omega-3 levels (P.001); (2) lengthening of ERPs by 8%-14% at all measured sites and pacing cycle lengths (P.05); (3) no effect on baseline interatrial, intra-atrial, left atrial, and coronary sinus conduction but a significant attenuation of maximal conduction delay (P.05); (4) less inducible AF (AF ≥30 seconds: 24.2% vs. 7.9%; P.001); (5) shorter mean duration of induced AF (P = .003); and (6) prolongation of induced AF cycle length (P.001).Chronic fish oil supplementation in humans prolongs atrial refractoriness and reduces vulnerability to inducible AF. These EP changes may explain the antifibrillatory effect of chronic fish oil ingestion.