Oxidized low-density lipoprotein (ox-LDL) contributes to the pathogenesis of atherosclerosis by promoting vascular endothelial cell injury.Gossypetin, a naturally occurring hexahydroxyflavone, has been shown to possess antimutagenic, antioxidant, antimicrobial, and antiatherosclerotic effects. In this study, the atheroprotective role of gossypetin was examined in endothelial cells. The protective effect of gossypetin against ox-LDL-induced injury in human umbilicalvein endothelial cells (HUVECs) was first noted at 0.1−0.5 μM. Gossypetin showed potential in reducing ox-LDL-dependent apoptosis, as demonstrated by morphological and biochemical features, including formation of apoptotic bodies,distribution of hypodiploid phase, and activation of caspase-3. Next, the ox-LDL induced formation of acidic vesicular organelles and the upregulation of autophagyrelated genes (LC3 and Beclin-1) were enhanced by gossypetin. Gossypetin triggered autophagic flux was further confirmed by an increase in the level of LC3-II under pretreatment conditions with an autophagy inhibitor, chloroquine (CQ). In addition, silencing Beclin-1 inhibited both the gossypetin-mediated protective affects and the autophagic process. Molecular data indicated that the autophagic effect of gossypetin might be mediated via the class III PI3K/Beclin-1 and PTEN/class I PI3K/Akt signaling cascades, as demonstrated by the use of a class III PI3K inhibitor, 3-methyladenine (3-MA), and a PTEN inhibitor, SF1670. Finally, gossypetin improved atherosclerotic lesions and endothelial injury in vivo. These data imply that gossypetin upregulates the autophagic pathway, which led to subsequent reduction of ox-LDL-induced atherogenic endothelial cell injury and apoptosis, and provide a new mechanism for the antiatherosclerotic activity of gossypetin.