Atherosclerosis is one of the underlying causes of cardiovascular disease. Formation of foam cells and necrotic core in the plaque is a hallmark of atherosclerosis, which results from lipid deposition, apoptosis, and inflammation in macrophage. Macrophage autophagy is a critical anti-atherogenic process and defective autophagy aggravates atherosclerosis by enhancing foam cell formation, apoptosis, and inflammation. Hence, enhancing autophagy can be a strategy for atherosclerosis treatment. Calycosin, a flavonoid from Astragali Radix, displays antioxidant and anti-inflammatory activities, and therefore is potential to reduce the risk of cardiovascular disease. However, the antiatherogenic effect of calycosin and the involved mechanism remains unclear. In this study, we assessed the potential benefits of calycosin on autophagy and atherosclerosis, and revealed the underlying mechanism.In this study, apoE-/- mice were fed high-fat diet for 16 weeks in presence of calycosin and/or autophagy inhibitor chloroquine, which was followed by determination of atherosclerosis development, autophagy activity, and the involved mechanisms.Calycosin protected against atherosclerosis and enhanced plaque stability via promoting autophagy. Calycosin inhibited foam cells formation, inflammation, and apoptosis by enhancing autophagy. MLKL was demonstrated as a new autophagy regulator, which can be negatively regulated by KLF2. Mechanistically, inhibitory effects of calycosin on atherogenesis were via improving autophagy through modulating KLF2-MLKL signaling pathway.This study demonstrated the atheroprotective effect of calycosin was through upregulating KLF2-MLKL-mediated autophagy, which not only proposed novel mechanistic insights into the atherogenesis but also identified calycosin as a potential drug candidate for atherosclerosis treatment.This article is protected by copyright. All rights reserved.