We aimed to investigate whether dietary intake of total or individual (n-3, n-6, and n-3:n-6 ratio) polyunsaturated fatty acids (PUFAs) was prospectively associated with serum levels of C-reactive protein (CRP), a marker of inflammation. We analyzed 4,707 participants (1,943 men and 2,764 women) from the Rotterdam Study, a prospective follow-up study of subjects aged 55 years or older in the Netherlands. At baseline (1989-1993), dietary intake of PUFAs was assessed by validated food frequency questionnaire. CRP was measured at baseline and at the third study visit (1997-1999). Regression coefficients (β) and 95% confidence intervals were obtained using linear generalized estimating equations. Dietary intake of butter and margarine explained most of the variance in PUFA intake. After adjustment for possible confounding factors, higher intake of total PUFAs was associated with lower CRP levels (fourth quartile vs. first quartile: β = -0.08, 95% confidence interval: -0.15, -0.01). Similarly, intake of n-6 PUFAs was inversely related to CRP (fourth quartile vs. first: β = -0.09, 95% confidence interval: -0.16, -0.01). No consistent trends were observed regarding n-3 PUFAs or n-3:n-6 PUFA ratio and CRP. These findings suggest that high intakes of total PUFAs are associated with lower levels of CRP, reflecting diminished chronic systemic inflammation, which in our study was mainly driven by n-6 PUFAs.