<p>Macrophages play differential roles in the pathogenesis of atherosclerosis due to their different phenotypes. Although α-SMA⁺ macrophages have been found to present in bone marrow and atherosclerotic plaques, their role in atherosclerosis remains unclear. By performing partial carotid ligation (PCL) on monocyte/macrophage lineage-tracked mice, we observed bone marrow-derived α-SMA⁺ macrophages in the subendothelium and atherosclerotic plaques under disturbed flow conditions. The functional role of α-SMA⁺ macrophages in atherosclerotic plaque formation was examined using macrophage-specific <i>Acta2</i> knockout (<i>Acta2</i>^MKO) mice generated by crossing <i>Acta2</i>^<i>f/f</i> transgenic mice with <i>LysM-Cre</i> mice. The size of the aortic plaques was 77.43% smaller in <i>Acta2</i>^<i>MKO</i> mice than in <i>Acta2</i>^<i>f/f</i> mice following adeno-associated virus-mutant <i>PCSK9</i> injection and high-fat diet (HFD) feeding for 12&#xa0;weeks. A significant reduction in lipid deposition, macrophage infiltration and the α-SMA⁺ area was observed in the aortic roots of <i>Acta2</i>^<i>MKO</i> mice compared with <i>Acta2</i>^<i>f/f</i> mice. Mechanistically, using <i>Acta2</i>-overexpressing Raw264.7 cells (<i>Acta2</i>^<i>hi</i> cells) and bone marrow-derived macrophages (BMDMs) from <i>Acta2</i>^<i>MKO</i> mice (<i>Acta2</i>^<i>MKO</i> BMDMs), we showed that macrophage α-SMA increased the expression of the scavenger receptor SR-A, induced Ox-LDL binding and uptake, and reduced the level of the cholesterol transporter ABCA1, potentially via the AKT pathway. Together, our results indicate that bone marrow-derived α-SMA⁺ macrophages contribute to atherosclerotic plaque formation due to dysregulated cholesterol uptake and efflux, providing potential targets for the prevention and treatment of atherosclerosis.</p>