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Background: Atherosclerosis is a chronic inflammatory disease. Although Toll-like receptor 4 (TLR4) has been involved in inflammatory atherosclerosis, the exact mechanisms by which oxidized-low-density lipoproteins (ox-LDL) activates TLR4 and elicits inflammatory genesis are not fully known. Myeloid differentiation factor 2 (MD2) is an extracellular molecule indispensable for lipopolysaccharide recognition of TLR4. Method: ApoE-/-MD2-/- mice and pharmacological inhibitor of MD2 were used in this study. We also reconstituted ApoE-/- mice with either ApoE-/- or ApoE-/-MD2-/- marrow-derived cells. Mechanistic studies were performed in primary macrophages, HEK-293T cells, and cell-free system. Finding: MD2 levels are elevated in atherosclerotic lesion macrophages, and MD2 deficiency or pharmacological inhibition in mice reduces the inflammation and stunts the development of atherosclerotic lesions in ApoE-/- mice fed with high-fat diet. Transfer of marrow-derived cells from ApoE-MD2 double knockout mice to ApoE knockout mice confirmed the critical role of macrophage-derived MD2 in inflammatory factor induction and atherosclerosis development. Mechanistically, we show that MD2 does not alter ox-LDL uptake by macrophages but is required for TLR4 activation and inflammation via directly binding to ox-LDL, which triggers MD2/TLR4 complex formation and TLR4-MyD88-NFκB pro-inflammatory cascade. Interpretation: We provide a mechanistic basis of ox-LDL-induced macrophage inflammation, illustrate the role of macrophage-derived MD2 in atherosclerosis, and support the therapeutic potential of MD2 targeting in atherosclerosis-driven cardiovascular diseases. Funding Statement: This work was supported by the National Key Research Project of China (2017YFA0506000), National Natural Science Foundation of China (21961142009, 81930108, 81670244, and 81700402), and Natural Science Foundation of Zhejiang Province (LY19H020004). Declaration of Interests: The authors have nothing to disclose. Ethics Approval Statement: All protocols involving animal experiments were approved by the Wenzhou Medical University Animal Policy and Welfare Committee and adhered to the NIH guidelines (Guide for the care and use of laboratory animals). All the procedures involving human samples were approved by the First Affiliated Hospital of Wenzhou Medical University in China and all samples were obtained with informed consent.