Rationale: We previously showed that early calcification of atherosclerotic plaques associates with macrophage accumulation. Chronic renal disease (CRD) and mineral imbalance accelerates calcification and the subsequent release of matrix vesicles (MVs) - precursors of microcalcification. Objective: We tested the hypothesis that macrophage-derived MVs contribute directly to microcalcification. Methods and Results: Macrophages associated with regions of calcified vesicular structures in human carotid plaques (n=136 patients). In vitro, macrophages released MVs with high calcification and aggregation potential. MVs expressed exosomal markers (CD9 and TSG101), and contained S100A9 and annexin V (Anx5). Silencing S100A9 in vitro and genetic deficiency in S100A9-/- mice reduced MV calcification, while stimulation with S100A9 increased calcification potential. Externalization of phosphatidylserine (PS) after Ca/P stimulation and interaction of S100A9 and Anx5, indicated that a PS-Anx5-S100A9 membrane complex facilitates hydroxyapatite nucleation within the macrophage-derived MV membrane. Conclusions: Our results support the novel concept that macrophages release calcifying MVs enriched in S100A9 and Anx5, which contribute to accelerated microcalcification in CRD.