Background <p>Severe disruption of lipid metabolism in vivo is one of the central mechanisms in the development of atherosclerotic vascular injury (AVI). Reverse cholesterol transport (RCT) plays a pivotal role in eliminating excess cholesterol, preventing lipid deposition in the aorta, and reducing plaque formation associated with AVI. Floralozone (FL) reduces endothelial cell injury in AVI rats by regulating sphingosine-1-phosphate (S1P) expression. However, FL’s potential to prevent AVI by modulating cholesterol metabolism remains unknown.</p> Methods <p>In this study, network pharmacology and molecular docking predicted FL’s potential targets in AVI protection. AVI rats were induced with a high-sugar, high-fat diet and vitamin D3 injection. FL intervention’s effects on aortic pathology and lipid levels were assessed. The expression levels of SREBP-1c, PPARγ, ABCA1, and ABCG1 were evaluated. Raw264.7 macrophages were induced to form foam cells with ox-LDL, and FL’s effects on the AMPKα/SREBP-1c pathway and miR-33-5p were investigated.</p> Results <p>FL reduced lipid levels and SREBP-1c expression, increased HDL-C, promoted ABCA1- and ABCG1-mediated cholesterol efflux, and reduced aortic cholesterol accumulation. The AMPKα inhibitor dorsomorphin blocked FL’s inhibition of intracellular cholesterol accumulation and SREBP1 down-regulation in foam cells. FL decreased miR-33-5p expression but up-regulated PPARγ, promoting ABCA1- and ABCG1-mediated cholesterol efflux. However, miR-33-5p mimic reduced FL-induced cholesterol efflux, while miR-33-5p inhibitor increased it.</p> Conclusion <p>FL may promote foam cell cholesterol efflux by modifying the AMPKα/SREBP-1c pathway and down-regulating miR-33-5p, which targets cholesterol metabolism genes (PPARγ, ABCA1, and ABCG1). These findings provide a new insight into the protective effect of FL on AVI.</p>