OBJECTIVES: A diet rich in fish and ω-3 polyunsaturated fatty acids (PUFAs) has been thought to reduce the risk for cardiovascular disease (CVD). The beneficial effects of fish oil and ω-3 PUFA on CVD can be mediated by epigenetic status of the genes associated with lipid metabolism and inflammation. The aim of this study was to investigate whether dietary fish and fatty acid (FA) intakes are associated with leukocyte ATP-binding cassette transporter A1 (ABCA1) DNA methylation levels in a Japanese population. METHODS: This cross-sectional study included 298 adults (137 men and 161 women) without clinical history of CVD or cancer. The pyrosequencing method was used to measure leukocyte ABCA1 DNA methylation levels. Dietary fish and FA intakes were assessed based on the validated food frequency questionnaire. RESULTS: Mean ABCA1 DNA methylation levels were significantly lower in the highest fish intake groups (≥5-6/wk) compared with the lowest intake group (≤1-2/wk; P = 0.004). In multivariable linear regression analyses, higher dietary intake of ω-3 PUFAs and ω-3 highly unsaturated fatty acids was significantly associated with decreased levels of ABCA1 DNA methylation (P = 0.001 and 0.005); whereas no significant associations were seen between intake of dietary saturated fatty acid, monounsaturated fatty acid, and ω-6 PUFAs and ABCA1 DNA methylation. CONCLUSION: Higher dietary fish and ω-3 PUFA intake were associated with lower ABCA1 DNA levels in a Japanese population. The present results may bring potential insights on biological mechanisms underlying the protective effects of dietary fish and ω-3 PUFA intakes on CVD.