Activation of retinoid X receptor (RXR) is known to exert antiatherogenic effects. However, the underlying mechanism remains unclear. In this study, we examined the effects of the RXR agonists 9-cis-retinoic acid and SR11237 on high-glucose-induced oxidative stress in human endothelial cells. Our results demonstrated that high-glucose-induced oxidative stress in human umbilical vein endothelial cells (HUVECs) was mainly mediated through its activation of the Nox4, gp91phox, and p22phox components of nicotinamide adenine dinucleotide phosphate oxidase. Treatment of endothelial cells with RXR agonists resulted in significant inhibition of high-glucose-induced oxidative stress and expression of Nox4, gp91phox, and p22phox. The effect of RXR agonists was due to their inhibition of Rac-1 activation. Furthermore, RXR agonists rapidly inhibited high-glucose-induced activation of protein kinase C (PKC), an upstream activator of Rac-1. To study whether the rapid inhibitory effects of RXR agonists were mediated by RXR, we examined the effect of RXR downregulation by RXR siRNA. Our results showed that expression of RXR siRNA largely abrogated the effects of RXR agonists, suggesting the requirement of RXR expression. Interestingly, RXRalpha, which was diffusely distributed in HUVECs, accumulated mainly in the nucleus upon high glucose exposure. Treatment of cells with RXR agonists prevented the effect of high glucose. Thus, RXR ligands rapidly inhibit high-glucose-induced oxidative stress by antagonizing high-glucose-induced PKC activation, and cytoplasmic RXRalpha is implicated in this regulation.