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Non-alcoholic fatty liver disease (NAFLD) is a common condition, in which abnormal amounts of triglycerides accumulate in hepatocytes and is closely related to cardiovascular diseases and diabetes. Dietary fats contribute 15% of fat accumulation in the liver and regulate hepatic lipid metabolism. The supplementation of n-3 polyunsaturated fatty acids (n-3 PUFAs) improves NAFLD. The aim of this study is to assess the cross-sectional association between dietary n-3 PUFAs and NAFLD in Japanese men and women.Participants were middle-aged, apparently healthy, 296 men and 496 women, who did not drink alcohol and who participated in a general health check-up program. Dietary information from the previous month was obtained by the brief-type self-administered diet history questionnaire. NAFLD was diagnosed if abdominal ultrasonography revealed the presence of fatty liver.The prevalence of NAFLD was 45.3% in men and 17.5% in women. In comparison with the first tertile, multivariate adjusted odds ratios (95% confidence intervals) for the presence of NAFLD in the second and third tertiles for men taking eicosapentaenoic acid (EPA) were 0.59 (0.31–1.14) and 0.45 (0.23–0.90), respectively, (P for linear trend=0.024), and the multivariate adjusted odds ratios (95% confidence intervals) for the presence of NAFLD in the second and third tertiles for men taking EPA+docosahexaenoic acid (DHA) were 0.44 (0.23–0.86) and 0.48 (0.24–0.95), respectively, (P for linear trend=0.035). However, there was no significant relation between NAFLD and each of these nutrients in women.Dietary EPA and EPA+DHA may be independent and preventive nutrients for NAFLD in Japanese men.