Dipeptidyl peptidase IV (DPP-IV) inhibitors are novel oral anti-hyperglycemic agents. Here, the anti-apoptotic effect of Anagliptin in human umbilical vein endothelial cells (HUVECs) was evaluated. Cultured HUVECs were pre-incubated with Anagliptin, and then treated hydrongen peroxide (H2O2) to induce apoptosis. The apoptosis of HUVECs were detected by viability, LIVE/DEAD staining assay and flow cytometry assays. HUVECs were transfected with plasmid harboring human NADPH oxidases (NOX) 4 or an empty vector. The formation of reactive oxygen species (ROS) was measured by immunofluorescence. Apoptotic and anti-apoptotic factor were detected by Western Blot. Pre-incubation with Anagliptin protected HUVECs from H2O2 induced apoptosis. The transfection assay also indicated that pre-incubation with Anagliptin inhibited the apoptosis of HUVECs induced by NADPH oxidase 4 (NOX-4) overexpression. Immunofluorescence demonstrated that pre-incubation with Anagliptin suppressed the formation of ROS in apoptotic HUVECs. Pre-incubation with Anagliptin inhibited NOX-4 mediated the Bax, caspase-3, cleave caspase-3 and Cyto C overexpression, but up-regulated the protein level of Bcl-2 in HUVECs. The data help us to better understand the effect of Anagliptin on apoptosis, and will be valuable in identifying new targets to prevent the endothelial cell apoptosis after injury.