Introduction: Evidence from previous studies suggests that dietary marine omega-3 fatty acids have cardioprotective properties. However, our understanding of the mechanism for these beneficial effects remains limited. We sought to examine the association between marine omega-3 fatty acid intake and coronary flow reserve (CFR), a measure of overall coronary vasodilator capacity and microvascular function. Hypothesis: Increased dietary intake of marine omega-3 fatty acids is associated with higher CFR. Methods: A sample of 259 male middle-aged twins, including 115 monozygotic and dizygotic twin pairs and 29 unpaired twins, with no previous history of coronary heart disease (CHD), was recruited from the Vietnam Era Twin Registry. Prior to the study visit, all twins completed a 3-day food record detailing their diet for the previous three days. Dietary information was analyzed using specialized nutrition software and reported intakes of various types of fish were used to derive marine omega-3 fatty acid (docosahexaenoic acid [DHA] and eicosapentaenoic acid [EPA]) intake. CFR was measured using positron emission tomography [N 13 ] ammonia with quantitation of myocardial blood flow at rest and after adenosine stress. Mixed-effect regression analysis was used to assess the association between marine omega-3 fatty acid intake and CFR both at the individual level and within twin pairs. Potential confounders included demographic characteristics, lifestyle factors (including physical activity), traditional cardiovascular disease (CVD) risk factors, use of omega-3 and multivitamin supplements, total energy intake, and other nutritional factors such as intake of cholesterol, sodium, alcohol, sugar-sweetened beverages, and saturated and monounsaturated fatty acids. Results: The twins’ mean age was 54 years (SD = 3.0) and 93.1% (241 out of 259) were white. An increase in dietary intake of marine omega-3 fatty acids of 1 g/d was associated with a 7.6% higher CFR (95% CI: 0.6, 15.2) after adjustment for potential confounders ( P = 0.033). This association also persisted within twin pairs: a 1 g/d within-pair difference in dietary intake of marine omega-3 fatty acids was associated with a 16.3% higher CFR in the twin with the greater dietary intake of marine omega-3 fatty acids than in the co-twin with the lower dietary intake ( P = 0.020). These associations did not differ by zygosity. Conclusions: Dietary intake of marine omega-3 fatty acids is directly associated with CFR, a measure of coronary microvascular function, independent of traditional CVD risk factors and shared familial and genetic factors. Our study suggests a potential novel mechanism for the protective effects of dietary marine omega-3 fatty acids on the cardiovascular system and supports the benefit of consuming marine omega-3 fatty acids to promote cardiovascular health.