Atherosclerosis is a chronic inflammation condition resulting from the interaction between lipoproteins, monocyte-derived macrophages, T lymphocytes, and other cellular elements in the arterial wall. Macrophage-derived foam cells play a key role in both early and advanced stage of atherosclerosis. Previous studies have shown that berberine could inhibit foam cell formation and prevent experimental atherosclerosis. However, its underlying molecular mechanisms have not been fully clarified. In this study, we explored the cholesterol-lowering effects of berberine in macrophage-derived foam cells and investigated its possible mechanisms in prevention and treatment of atherosclerosis. Here, we demonstrated that berberine could inhibit atherosclerosis in apolipoprotein E-deficient mice and induce cholesterol reduction as well as decrease the content of macrophages. Berberine can regulate oxLDL uptake and cholesterol efflux, thus suppresses foam cell formation. Mechanisms study showed that berberine can suppress scavenger receptor expression via inhibiting the activity of AP-1 and upregulate ATP-binding cassette transporter via activating Nrf2/HO-1 signaling in human macrophage. In summary, berberine significantly inhibits atherosclerotic disease development by regulating lipid homeostasis and suppressing macrophage foam cell formation.