Vascular oxidative stress has been found to be a prominent feature underlying the vascular abnormalities of patients with insulin-resistance and type-2 diabetes. Short chain fatty acids (SCFA) i.e. acetate (NaA), propionate and butyrate are by-products of resistant starch fermentation by anaerobic bacteria in the intestine. SCFA have been shown to be anti-inflammatory against vascular oxidative stress, but the molecular mechanism of SCFA action remains unknown. GPCR43 belongs to a family of orphan G-protein coupled receptor (GPCR) and is a newly discovered receptor of SCFA. In this study we investigated the relationship between high glucose-induced oxidative stress and the levels of expression of GPCR43 in cultured human pulmonary microvascular endothelial cells (HPMEC) and human saphenous vein segments (HSV) in the presence or absence of NaA (5 mM), a reported agonist of GPCR43. Compared to the control endothelial cells or HSV cultured in the normal glucose (5 mM) medium, high glucose (24 h) significantly increased reactive oxygen species (ROS) production by both HPMEC (48.5 ± 12.6%; p < 0.05) and HSV as measured by lucigenin (5 mM)-chemiluminescence and DHE fluorescence. Increased ROS production was accompanied by significant increases in NADPH oxidase 2 (Nox2) and p53. GPCR43 was detected in HPMEC as shown by immunofluorescence flow cytometry, and the endothelium and media of HSV as shown by confocal microscopy and immuno-histo-chemistry. NaA itself (used in normal glucose medium) increased the levels of GPCR43 expression without significant effect on vascular ROS production and Nox2 expression. However, when used together with high glucose, NaA attenuated significantly the high glucose-induced ROS production by both HPMEC and HSV and inhibited endothelial apoptosis. In conclusion, GPCR43 plays an important role in maintaining vascular redox-homeostasis under high glucose challenge. GPCR43 agonists such as acetate may have the therapeutic potential for treating oxidative stress-related vascular complications in patients with insulin-resistance and type-2 diabetes.