Objective- Inflammatory stimuli enhance the progression of atherosclerotic disease. Inflammation also increases the expression of hepcidin, a hormonal regulator of iron homeostasis, which decreases intestinal iron absorption, reduces serum iron levels and traps iron within macrophages. The role of macrophage iron in the development of atherosclerosis remains incompletely understood. The objective of this study was to investigate the effects of hepcidin deficiency and decreased macrophage iron on the development of atherosclerosis. Approach and Results- Hepcidin- and LDL (low-density lipoprotein) receptor-deficient ( Hamp/ Ldlr) mice and Hamp/ Ldlr control mice were fed a high-fat diet for 21 weeks. Compared with control mice, Hamp/ Ldlr mice had decreased aortic macrophage activity and atherosclerosis. Because hepcidin deficiency is associated with both increased serum iron and decreased macrophage iron, the possibility that increased serum iron was responsible for decreased atherosclerosis in Hamp/ Ldlr mice was considered. Hamp/ Ldlr mice were treated with iron dextran so as to produce a 2-fold increase in serum iron. Increased serum iron did not decrease atherosclerosis in Hamp/ Ldlr mice. Aortic macrophages from Hamp/ Ldlr mice had less labile free iron and exhibited a reduced proinflammatory (M1) phenotype compared with macrophages from Hamp/ Ldlr mice. THP1 human macrophages treated with an iron chelator were used to model hepcidin deficiency in vitro. Treatment with an iron chelator reduced LPS (lipopolysaccharide)-induced M1 phenotypic expression and decreased uptake of oxidized LDL. Conclusions- In summary, in a hyperlipidemic mouse model, hepcidin deficiency was associated with decreased macrophage iron, a reduced aortic macrophage inflammatory phenotype and protection from atherosclerosis. The results indicate that decreasing hepcidin activity, with the resulting decrease in macrophage iron, may prove to be a novel strategy for the treatment of atherosclerosis.