The study has been designed to investigate the effect of demethylasterroquinone B1 (DAQ B1), an activator of Akt, in diabetes mellitus (DM) and hyperhomocyteinemia (HHcy)-induced vascular endothelial dysfunction. Streptozotocin (55 mg kg⁻¹, i.v.) and methionine (1.7% w/w, p.o., 4 weeks) were administered to rats to produce DM (serum glucose >140 mg dl⁻¹) and HHcy (serum homocysteine >10 µM), respectively. Vascular endothelial dysfunction was assessed using isolated aortic ring preparation, electron microscopy of thoracic aorta and serum concentration of nitrite/nitrate. The expression of messenger RNA for p22phox and eNOS was assessed by reverse transcription-polymerase chain reaction. Serum thiobarbituric acid reactive substances (TBARS) and aortic superoxide anion were estimated to assess oxidative stress. DAQ B1 (5 mg kg⁻¹, p.o.) or atorvastatin (30 mg kg⁻¹, p.o.) in diabetic and hyperhomocysteinemic rats significantly reduced serum glucose and homocysteine concentration. DAQ B1 or atorvastatin markedly improved acetylcholine-induced endothelium-dependent relaxation, vascular endothelial lining, serum nitrite/nitrate concentration and serum TBARS in diabetic and hyperhomocysteinemic rats. However, this ameliorative effect of DAQ B1 has been prevented by L-NAME (25 mg kg⁻¹, i.p.), an inhibitor of eNOS. Therefore, it may be concluded that DAQ B1-induced activation of Akt may activate eNOS and consequently reduce oxidative stress to improve vascular endothelial dysfunction.