We evaluated the hypothesis that intake of (n-3) fatty acids is inversely associated with biomarkers of inflammation and endothelial activation. We conducted a cross-sectional study of 727 women from the Nurses’ Health Study I cohort, aged 43–69 y, apparently healthy at time of a blood draw in 1990. Dietary intake was assessed by a validated FFQ in 1986 and 1990. C-reactive protein (CRP) levels were 29% lower among those in the highest quintile of total (n-3) fatty acids, compared with the lowest quintile; interleukin-6 (IL-6) levels were 23% lower, E-selectin levels 10% lower, soluble intracellular adhesion molecule (sICAM-1) levels 7% lower, and soluble vascular adhesion molecule (sVCAM-1) levels 8% lower. The intake of α-linolenic acid was inversely related to plasma concentrations of CRP (β = −0.55, P = 0.02), Il-6 (β = −0.36, P = 0.01), and E-selectin (β = −0.24, P = 0.008) after controlling for age, BMI, physical activity, smoking status, alcohol consumption, and intake of linoleic acid (n-6) and saturated fat. Long-chain (n-3) fatty acids (eicosapentaenoic and docosahexaenoic) were inversely related to sICAM-1 (β = −0.11, P = 0.03) and sVCAM-1 (β = −0.17, P = 0.003). Total (n-3) fatty acids had an inverse relation with CRP (β = −0.44, P = 0.007), IL-6 (β = −0.26, P = 0.009), E-selectin (β = −0.17, P = 0.004), sICAM-1 (β = −0.07, P = 0.02), and sVCAM-1 (β = −0.10, P = 0.004). These associations were not modified by intake of vitamin E, dietary fiber, trans fatty acids, or by the use of postmenopausal hormone therapy. In conclusion, this study suggests that dietary (n-3) fatty acids are associated with levels of these biomarkers reflecting lower levels of inflammation and endothelial activation, which might explain in part the effect of these fatty acids in preventing cardiovascular disease.