Inflammatory damage to endothelial cells plays a pivotal role in the diabetes-provoked atherosclerosis (AS). PYD domains-containing protein 3 (NLRP3) induces formation of inflammasome activates caspase-1, which subsequently cleaves the precursor form of IL-1β (pro-IL-1β) into the processed, secreted form IL-1β to promote the immune responses in AS. However, it is not known whether NLRP3 activation specifically in endothelial cells causes AS. Here, in an in vitro model for AS, we showed that NLRP3-depleted human aortic endothelial cells (HAECs) became resistant to apoptotic cell death, maintained proliferative potential and reduced reactive oxygen species (ROS) production upon treatment with oxidized low-density lipoprotein (ox-LDL). Next, the role of NLRP3 in endothelial cells in the development of diabetes-associated AS was assessed in endothelial cell-specific NLRP3 mutant, ApoE (-/-) mice (APOEKO/Tie2p-Cre/NLRP3MKO), compared to control ApoE (-/-) mice (APOEKO), supplied with either high-fat diet (HFD), or normal diet (ND). We found that endothelia-specific NLRP3-depletion significantly attenuated AS severity in mice treated with HFD, likely through reduced apoptotic death of endothelial cells and production of ROS. Together, our data suggest that NLRP3 activation in endothelial cells promotes development of diabetes-associated AS.