Few studies have examined the association of long-chain n-3 polyunsaturated fatty acids (LCn-3PUFAs) with the measures of atherosclerosis in the general population. This study aimed to examine the relationship of total LCn-3PUFAs, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) with aortic calcification. In a multiethnic population-based cross-sectional study of 998 asymptomatic men aged 40–49 years (300 US-White, 101 US-Black, 287 Japanese American, and 310 Japanese in Japan), we examined the relationship of serum LCn-3PUFAs to aortic calcification (measured by electron-beam computed tomography and quantified using the Agatston method) using Tobit regression and ordinal logistic regression after adjusting for potential confounders. Overall 56.5% participants had an aortic calcification score (AoCaS) > 0. The means (SD) of total LCn-3PUFAs, EPA, and DHA were 5.8% (3.3%), 1.4% (1.3%), and 3.7% (2.1%), respectively. In multivariable-adjusted Tobit regression, a 1-SD increase in total LCn-3PUFAs, EPA, and DHA was associated with 29% (95% CI = 0.51, 1.00), 9% (95% CI = 0.68, 1.23), and 35% (95% CI = 0.46, 0.91) lower AoCaS, respectively. Results were similar in ordinal logistic regression analysis. There was no significant interaction between race/ethnicity and total LCn–3PUFAs, EPA or DHA on aortic calcification. This study showed the significant inverse association of LCn-3PUFAs with aortic calcification independent of conventional cardiovascular risk factors among men in the general population. This association appeared to be driven by DHA but not EPA. •Evidence concerning LCn-3PUFAs and atherosclerosis is limited in the general population.•Cross-sectionally examined the relationship of LCn-3 PUFAs to aortic calcification in asymptomatic middle aged men.•Overall, LCn-3PUFAs especially DHA was inversely and significantly associated with aortic calcification.•No significant interaction between race/ethnicity and total LCn-3 PUFAs, EPA or DHA on aortic calcification.•In the general middle-aged male population, DHA may be more anti-atherogenic than EPA.