Methylglyoxal (MGO) is a reactive dicarbonyl metabolite of glucose, and its plasma levels are elevated in patients with diabetes. Studies have shown that MGO combines with the amino and sulphhydryl groups of proteins to form stable advanced glycation end products (AGEs), which are associated with vascular endothelial cell (EC) injury and may contribute to the progression of atherosclerosis. In this study, MGO induced apoptosis in a dose-dependent manner in HUVECs, which was attenuated by pre-treatment with z-VAD, a pan caspase inhibitor. Treatment with MGO increased ROS levels, followed by dose-dependent down-regulation of c-FLIP . In addition, pre-treatment with the ROS scavenger NAC prevented the MGO-induced down-regulation of p65 and c-FLIP , and the forced expression of c-FLIP attenuated MGO-mediated apoptosis. Furthermore, MGO-induced apoptotic cell death in endothelium isolated from mouse aortas. Finally, MGO was found to induce apoptosis by down-regulating p65 expression at both the transcriptional and posttranslational levels, and thus, to inhibit c-FLIP mRNA expression by suppressing NF-κB transcriptional activity. Collectively, this study showed that MGO-induced apoptosis is dependent on c-FLIP down-regulation via ROS-mediated down-regulation of p65 expression in endothelial cells.