The cardioprotective effects of long-chain n-3 polyunsaturated fatty acids (PUFAs) and fish consumption have been observed. However, data on the specific associations of these dietary factors with inflammation and endothelial activation are sparse. A cross-sectional study was conducted of 5,677 men and women from the Multi-Ethnic Study of Atherosclerosis (MESA) cohort, including African Americans, Caucasians, Chinese, and Hispanics aged 45 to 84 years and free of clinical cardiovascular disease. Dietary information was collected using a self-administered food frequency questionnaire. Multivariate linear regression analyses were used to examine relations between the intake of long-chain n-3 PUFAs, nonfried fish, and fried fish and biomarkers of inflammation and endothelial activation. Long-chain n-3 PUFA intake was inversely associated with plasma concentrations of interleukin-6 (p = 0.01) and matrix metalloproteinase-3 (p = 0.03) independent of age, body mass index, physical activity, smoking, alcohol consumption, and dietary variables. Nonfried fish consumption was inversely related to C-reactive protein (p = 0.045) and interleukin-6 (p <0.01), and fried fish consumption was inversely related to soluble intercellular adhesion molecule-1 (p <0.01) but was not associated with other biomarkers after adjustment for potential confounders. In conclusion, the results of this study suggest that the dietary intake of long-chain n-3 PUFAs and fish is inversely associated with concentrations of some biomarkers, reflecting lower levels of inflammation and endothelial activation. These results may partially explain the cardioprotective effects of fish consumption.