Abstract Body (Do not enter title and authors here): Iron retention is commonly observed in atherosclerotic plaques and is believed to be detrimental to atherosclerosis. P2Y12 expressed in platelets is a target of antiplatelet therapy in preventing thrombotic complications of atherosclerosis. Our previous work discovered the protective effect of zebrafish P2Y12 on hematopoiesis, implying the involvement of P2Y12 in iron metabolism. To verify this hypothesis, we focused on the role of P2Y12 in the iron metabolism of macrophages, the key player in systemic iron homeostasis and atherosclerosis. The case-control study showed that increased levels of serum iron were positively associated with the use of P2Y12 inhibitors, with an odds ratio (OR) of 10.333 (1.281-83.370). Elevated serum iron levels and transferrin saturation, reduced hepatic and splenic iron content, and decreased iron-associated Prussian blue staining in macrophages were observed in P2Y12-deficient zebrafish fed a two-week high-iron diet. Additionally, deficiency of P2Y12 in ApoEb^-/- zebrafish fed a high-fat diet reduced atherosclerosis progression and intraplaque iron retention. Furthermore, reduced expression of ferritin, restored cell viability and expression of ferroptosis marker proteins, and decreased ROS formation and inflammatory cytokines were observed in both iron-overloaded murine primary peritoneal macrophages (PMs) and ox-LDL-treated PMs with P2Y12 knockdown in vitro, while reversed phenotypes were observed after agonist-induced P2Y12 activation. Mechanistically, P2Y12 inhibition in iron-overloaded or ox-LDL-treated PMs suppressed the phosphorylation of NF-κB p65 and the expression of hepcidin, both of which were reversed by P2Y12 activation. In summary, P2Y12 inhibition decreased hepcidin autocrine through repressing NF-κB p65 phosphorylation in macrophages, preventing intracellular iron retention and atherosclerosis. These results identify macrophage P2Y12 as a potential therapeutic target for iron overload and atherosclerosis and indicate a possible side effect of P2Y12 inhibitors, which might help individualize the strategy of antiplatelet therapy.