Endothelial cell (EC) dysfunction is a key event in the onset and progression of atherosclerosis. Apoptosis may lead to endothelial dysfunction and contribute to vascular complications. Cyclophilin A (CyPA) is the main reactive oxygen species-induced factor that enhances the inflammatory activity of vascular cells in atherosclerotic plaques. However, the mechanism by which CyPA induces EC apoptosis is not entirely clear. Through Western blot, it demonstrated that extracellular CyPA activated the Akt and NF-κB pathway, followed by the upregulation of antiapoptotic protein Bcl-2 expression in ECs. When blocking intracellular CyPA by small interfering RNA in ECs, the effects of TNF-α-induced EC apoptosis and proapoptotic protein caspase-3 expression were significantly inhibited. This study shows that CyPA may initiate antiapoptotic and proapoptotic signaling in ECs, especially in response to reactive oxygen species stimulation. It serves as a potential target for atherosclerosis therapy.