Objective— Iron and the iron regulatory hormone hepcidin, major determinant of body iron distribution, are hypothesized to play a role in cardiovascular disease. Here, we assess the associations of hepcidin as well as ferritin, iron, total iron-binding capacity, and transferrin saturation (ie, iron parameters) with noninvasive measurements of atherosclerosis in men and women of a population-based cohort. Approach and Results— We included 766 participants of the Nijmegen Biomedical Study aged 46 to 67 years for whom serum measurements of hepcidin, iron parameters, and noninvasive measurements of atherosclerosis were available. Noninvasive measurements of atherosclerosis were presence of plaque, ankle-brachial index, and intima-media thickness. We performed multivariable logistic and linear regression analyses using quartiles of hepcidin and iron parameters. Analyses were stratified by sex and adjusted for several demographic, clinical, and biochemical determinants, including traditional risk factors of cardiovascular disease based on the Framingham risk score. Hepcidin and the hepcidin/ferritin ratio, reflecting hepcidin expression relative to iron stores, were significantly associated with the presence of plaque in women (adjusted odds ratios for quartile 4 versus quartile 1 [95% confidence intervals] of 3.07 [1.36–6.90] and 2.31 [1.03–5.18], respectively). The hepcidin/ferritin ratio was significantly and negatively associated with ankle-brachial index at rest in men and women (adjusted β for quartile 4 versus quartile 1 [95% confidence intervals] of −0.03 [−0.07 to 0.00] and −0.04 [−0.06 to −0.01], respectively). Conclusions— Our results suggest that the body iron distribution as determined by hepcidin affects the development of atherosclerosis in women.