We have previously reported that antioxidant response element (ARE)-regulated genes, such as heme oxygenase 1 (HO-1), sequestosome 1 (SQSTM1), and NAD(P)H quinone oxidoreductase 1 (NQO1), are induced in human umbilical vein endothelial cells (HUVEC) upon exposure to laminar shear stress. In the present study, we have confirmed a critical role for NF-E2-related factor 2 (Nrf2) in the induction of gene expression in HUVEC exposed to laminar shear stress. Although the mRNA levels of Nrf2 were unchanged during exposure to shear stress, the protein levels of Nrf2 were markedly increased. Small interfering RNA (SiRNA) against Nrf2 significantly attenuated the expression of Nrf2-regulated genes such as HO-1, SQSTM1, NQO1, glutamate-cysteine ligase modifier subunit (GCLM), and ferritin heavy chain. Nrf2 was rapidly degraded in cells treated with cycloheximide under static conditions, but shear stress decreased the rate of Nrf2 degradation. Incubation with the thiol antioxidant N-acetylcysteine strongly inhibited both the Nrf2 accumulation and the expression of Nrf2-regulated genes such as HO-1, GCLM, and SQSTM1. Nitric oxide (NO) production was increased with the strength of shear stress but neither the inhibitor of endothelial NO synthase (eNOS) nor the siRNA against eNOS affected the expression of Nrf2-regulated genes. A xanthine oxidase inhibitor oxypurinol and the flavoprotein inhibitor diphenyleneiodonium, which inhibits NAD(P)H oxidase and mitochondrial respiratory chain, markedly suppressed the expression of these genes. Moreover, diphenylpyrenlphosphine, a reducing compound of lipid hydroperoxides, also significantly suppressed Nrf2-regulated gene expression. Taken together, these findings suggest that shear stress stabilizes Nrf2 protein via the lipid peroxidation elicited by xanthine oxidase and flavoprotein mediated generation of superoxide, resulting in gene induction by the Nrf2-ARE signaling pathway.