Docosahexaenoic acid (DHA) and arachidonic acid (ARA) are the predominant long-chain polyunsaturated fatty acids (PUFA) among membrane phospholipids in the mammalian brain and neural tissues. This cross-sectional study examined age effects on serum eicosapentaenoic acid (EPA), DHA, and ARA compositions assessed with reference to dietary intakes among 1,014 Japanese men and 1,028 Japanese women aged 40-79 years. Venous blood was collected early in the morning after at least 12-h fasting. Serum fatty acid (FA) compositions were expressed as molar percentages of the total FA (mol% of total). Diet was assessed using a 3-day dietary record that included photographs. Participants were categorized into groups by sex and age (40-49, 50-59, 60-69, and 70-79 years). Intakes of fish, EPA, and DHA tended to increase with age. Significant positive correlations between serum FA composition and the corresponding weight percentage of total FA intake were observed for EPA and DHA in all sex and age groups, and for ARA among females in their 40s. Serum EPA and DHA compositions were higher, while ARA decreased with age, and these associations remained consistent even after adjusting for corresponding FA intake. These results suggest potential effects of age on differences in blood EPA, DHA, and ARA compositions, independent of corresponding FA intake among community-dwelling Japanese men and women.