Glycogen is an important source of energy production during endurance exercise, such as marathon. Due to limited storage of glycogen in muscle and liver, augmentation of fat oxidation is known to delay depletion of muscle glycogen, leading to improvement of endurance performance. N-3 polyunsaturated fatty acids (n-3 PUFA) resulting from the form of fish has shown to enhance fat oxidation at rest. However, effect of n-3 PUFA on substrate metabolism during prolonged exercise remains unclear. The present study was designed to investigate whether dietary n-3 PUFA enhances fat oxidation during exercise and endurance performance. Thirteen healthy men were divided into n-3 PUFA group [n=6, 6g/day of fish oil; 1,800mg eicosapentaenoic acid (EPA), 2,700mg docosahexiaenoic acid (DHA)] or CON group [n=7, 6g/day of olive oil]. The subjects ingested each supplement for 4-weeks. Before and after the treatment period, a 60-min pedaling exercise at 65% of maximal oxygen uptake followed by 5 km-time-trial was conducted. Resting concentrations of serum EPA and DHA, EPA/AA were significantly elevated in the n-3 PUFA group only. After supplementation period, the n-3 PUFA group increased significantly exercise-induced elevations of serum free fatty acids and glycerol concentrations, and lowered respiratory exchange ratio during a 60-min pedaling exercise. Similar changes were not observed in the CON group. However, treatment with n-3 PUFA did not affect significantly result of 5km-time-trial. Four-week supplementation of n-3 PUFA increased exercise-induced lipolysis and fat oxidation during prolonged exercise. However, the augmented fat metabolism did not affect endurance performance.