AbstractAtherosclerosis is one of the leading causes of morbidity and mortality, mainly due to the immune response triggered by the recruitment of monocytes/macrophages in the artery wall. Accumulating evidence have shown that matrix stiffness and oxidized low‐density lipoproteins (ox‐LDL) play important roles in atherosclerosis through modulating cellular behaviors. However, whether there is a synergistic effect for ox‐LDL and matrix stiffness on macrophages behavior has not been explored yet. In this study, we developed a model system to investigate the synergistic role of ox‐LDL and matrix stiffness on macrophage behaviors, such as migration, inflammatory and apoptosis. We found that there was a matrix stiffness‐dependent behavior of monocyte‐derived macrophages stimulated with ox‐LDL. What's more, macrophages were more sensitive to ox‐LDL on the stiff matrices compared to cells cultured on the soft matrices. Through next‐generation sequencing, we identified miRNAs in response to matrix stiffness and ox‐LDL and predicted pathways that showed the capability of miRNAs in directing macrophages fates. Our study provides a novel understanding of the important synergistic role of ox‐LDL and matrix stiffness in modulating macrophages behaviors, especially through miRNAs signaling pathways, which could be potential key regulators in atherosclerosis and immune‐targeted therapies.