Oxidized low-density lipoprotein (ox-LDL)-associated endothelial dysfunction is a critical factor in the initiation and progression of Atherosclerosis (AS). Annexin A1 is an important member of the annexin family. Despite its wide range of biological functions across various tissues and cells, the role of Annexin A1 in AS remains largely unexplored. In this study, we demonstrate that Annexin A1 treatment effectively reduced the expression of LOX-1 at both the mRNA and protein levels in HUVECs exposed to ox-LDL. Annexin A1 also ameliorated oxidative stress (OS) by decreasing mitochondrial ROS levels and restoring reduced GSH levels. Moreover, Annexin A1 decreased the expression of pro-inflammatory cytokines, including IL-6 and MCP-1. Importantly, Annexin A1 inhibited ox-LDL-induced expressions of the endothelial adhesion molecules, such as E-selectin and VCAM-1 in HUVECs, which leads to reduced attachment of THP-1 monocytes to HUVECs. Mechanically, we found that Annexin A1 reversed the expression of KLF2 against ox-LDL mediated by the PI3K/Akt axis. Notably, the silencing of KLF2 abrogated the protective effects of Annexin A1 on E-selectin and VCAM-1 expression and the attachment of THP-1 monocytes to HUVECs. Our findings suggest that Annexin A1 is a potential therapeutic agent for atherosclerosis, offering a novel approach to mitigate endothelial dysfunction and inflammation.© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.