Background— High intake of polyunsaturated fatty acids (PUFAs) may reduce the risk of cardiovascular disease (CVD) and mortality. Large, prospective studies including both sexes and circulating PUFAs as dietary biomarkers are needed. We investigated sex-specific associations of the major dietary PUFAs, eicosapentaenoic acid, docohexaenoic acid, linoleic acid, and α-linolenic acid, with incident CVD and all-cause mortality in a population-based cohort. Methods and Results— PUFAs in serum cholesterol esters were measured at baseline in 60-year-old Swedish women (n=2193) and men (n=2039). With the use of national registers, 484 incident CVD events (294 men and 190 women) and 456 all-cause deaths (265 men and 191 women) were identified during follow-up (median, 14.5 years) in individuals without prior CVD at baseline. Associations of PUFAs with CVD and mortality were evaluated with Cox proportional hazard models. In multivariable-adjusted models, 1-SD increases in eicosapentaenoic acid and docohexaenoic acid were associated with lower risk of incident CVD among women (hazard ratio [HR], 0.79 [95% confidence interval (CI), 0.64–0.97] and 0.74 [95% CI, 0.61–0.89], respectively). α-Linolenic acid was associated with moderately increased CVD risk in women (HR, 1.16; 95% CI, 1.02–1.32). Inverse associations with all-cause mortality were observed for eicosapentaenoic acid and docohexaenoic acid among all participants (HR, 0.81 [95% CI, 0.72–0.91] and 0.80 [95% CI, 0.72–0.89], respectively) and for linoleic acid in men (HR, 0.73; 95% CI, 0.64–0.83). Conclusions— Serum linoleic acid and very-long-chain n-3 PUFAs, partly reflecting vegetable oil and fish intake, respectively, were inversely associated with all-cause mortality. Inverse associations of eicosapentaenoic acid and docohexaenoic acid with incident CVD were observed only in women.