High glucose induces vascular smooth muscle cell (SMC) dysfunction by generating oxidative stress attributable, in part, to the up-regulated NADPH oxidases (Nox). We have attempted to elucidate the high-glucose-generated molecular signals that mediate this effect and hypothesize that products of high-glucose-induced lipid peroxidation regulate Nox by activating peroxisome proliferator-activated receptors (PPARs). Human aortic SMCs were exposed to glucose (5.5-25 mM) or 4-hydroxynonenal (1-25 μM, 4-HNE). Lucigenin assay, real-time polymerase chain reaction, western blot, and promoter analyses were employed to investigate Nox. We found that high glucose generated an increase in Nox activity and expression. It also promoted oxidative stress that consequently induced lipid peroxidation, which resulted in the production of 4-HNE. Pharmacological inhibition of Nox activity significantly reduced the formation of high-glucose-induced 4-HNE. Exposure of SMCs to non-cytotoxic concentrations (1-10 μM) of 4-HNE alone mimicked the effect of high glucose incubation, whereas scavenging of 4-HNE by N-acetyl L-cysteine completely abolished both the effects of high glucose and 4-HNE. The latter exerted its effect by activating PPARα and PPARβ/δ, but not PPARγ, as assessed pharmacologically by the inhibitory effect of selective antagonists and following the silencing of the expression of these receptors. These new data indicate that 4-HNE, generated following Nox activation, functions as an endogenous activator of PPARα and PPARβ/δ. The newly discovered "lipid peroxidation products-PPARs-Nox axis" represents a novel mechanism of Nox regulation and an additional therapeutic target for oxidative stress in diabetes.