Abstract We investigated the effect of high levels of n−3 highly unsaturated fatty acids (n−3 HUFA) in broodstock diet on egg quality and chemical composition of eggs of Japanese flounder. The broodstock were fed diets containing three levels of n−3 HUFA (2.1%, 4.8% or 6.2%) 2 months before and during the spawning period. No significant difference was found for weight gain of broodstock among the treatments. Egg production was highest in fish fed the highest level of n−3 HUFA. However, egg quality parameters, such as percentage of buoyant eggs, hatching rate and percentage of normal larvae, were significantly higher in the group fed the lowest n−3 HUFA diet. The fatty acid composition of eggs was influenced more markedly in the neutral lipid fraction than in the polar lipid fraction by dietary n−3 HUFA levels. Arachidonic acid (AA; 20:4n−6) and egg quality parameters both decreased with increasing dietary n−3 HUFA levels. The results suggest that a high level of n−3 HUFA in broodstock diet negatively affects egg quality of Japanese flounder.