Background <p>Atherosclerosis (AS) is a chronic arterial disease. Macrophage ferroptosis, a form of iron-dependent cell death, contributes significantly to plaque formation. Acyl-CoA synthetase long-chain family member 6 (ACSL6) is associated with ferroptosis, but its regulatory role in AS is currently unknown. This study aimed to investigate the function and mechanism of ACSL6 in AS.</p> Methods <p>We established an AS model by feeding apolipoprotein E-deficient (ApoE^−/−) mice a high-fat diet. ACSL6 expression was knocked down via tail vein injection of adeno-associated virus carrying ACSL6 short hairpin RNA (AAV-shACSL6). Subsequent changes in aortic pathology, ferroptosis-related markers, and nuclear factor erythroid-related factor 2 (Nrf2) expression were examined. In vitro, oxidized low-density lipoprotein (ox-LDL)-treated macrophages were constructed as a cell model and transfected with shACSL6 to inhibit ACSL6, and macrophage ferroptosis levels and Nrf2 activation were explored.</p> Results <p>ACSL6 expression levels were elevated in both in vitro and in vivo AS models. Expression levels of glutathione peroxidase 4 (GPX4) and Xap5 circadian timekeeper (xCT) were decreased in AS. Immunofluorescence (IF) confirmed infiltration of CD68-positive macrophages and reduced GPX4 expression in the aortas of AS mice. Nrf2 nuclear translocation was inhibited. Knockdown of ACSL6 reversed these changes, while co-treatment with the Nrf2 inhibitor ML385 blocked the protective effects of ACSL6 knockdown.</p> Conclusion <p>In conclusion, our results demonstrate that ACSL6 inhibition ameliorates AS by suppressing macrophage ferroptosis, a process in which the activation of the Nrf2 pathway plays a pivotal role. Our findings uncover a novel regulatory axis and provide new insights for the treatment of AS.</p>