Aim Vaspin (visceral adipose tissue‐derived serine protease inhibitor) is a novel adipocytokine found in visceral white adipose tissues of obese type 2 diabetic rats. We have previously shown that vaspin has anti‐inflammatory and antimigratory effects in vascular smooth muscle cells. Methylglyoxal (MGO) is an active metabolite of glucose and mediates diabetic vascular complications including endothelial cell (EC) apoptosis. Nonetheless, effects of vaspin on MGO‐induced apoptosis of vascular EC remain to be determined. We investigated the effects of vaspin on MGO‐induced apoptosis of human umbilical vein ECs (HUVECs). Methods Human umbilical vein ECs were treated with MGO (560 μm, 12 h) in the absence or presence of vaspin (1 ng mL−1, pre‐treatment for 2 h). Cell death was evaluated by a cell counting assay. Apoptosis was determined by a terminal deoxyribonucleotide transferase‐mediated deoxyuridine triphosphate nick‐end labelling (TUNEL) assay. Cleaved caspase‐3 expression was determined by Western blotting. Reactive oxygen species (ROS) generation was fluorometrically measured using 2′, 7′‐dichlorodihydrofluorescein diacetate. NADPH oxidase (NOX) activity was determined by a lucigenin assay. Results Vaspin significantly inhibited MGO‐induced HUVEC death. Vaspin significantly attenuated MGO‐increased TUNEL‐positive ECs. Moreover, vaspin significantly inhibited MGO‐induced caspase‐3 cleavage. Vaspin significantly inhibited MGO‐induced ROS generation as well as NOX activation. Conclusions The present results for the first time demonstrate that vaspin inhibits MGO‐induced EC apoptosis by preventing caspase‐3 activation via the inhibition of NOX‐derived ROS generation.