Atherosclerosis is often observed in areas where disturbed flow is formed, whereas atheroprotective region is found in areas where steady laminar flow is developed. It has been reported that some genes activated by blood flow play important roles in vascular function and pathogenesis of atherosclerosis. Extracellular signal-regulated kinase 5 (ERK5) has been reported to regulate endothelial integrity and protect from vascular dysfunction and disease under laminar flow. Krüppel-like factor 2 (KLF2) and NF-E2-related factor 2 (Nrf2) are major transcriptional factors that contribute to anti-atherogenic responses under laminar flow. Implication of ERK5 in laminar flow-mediated regulation of KLF2-dependent gene has been established, whereas the role of ERK5 in laminar flow-mediated activation of Nrf2 pathway has not been addressed yet. In this study, we found that the blockage of ERK5 either by genetic depletion with siRNA or by biochemical inactivation with a specific chemical compound inhibited laminar flow-induced upregulation of Nrf2-dependent gene expressions, whereas activation of ERK5 increased transcriptional activity and nuclear translocation of Nrf2, which suggests that ERK5 mediates laminar flow-induced up-regulation of Nrf2-dependent gene expression. Further functional studies showed that ERK5 provides protection against oxidative stress-induced cytotoxicity dependent on Nrf2. Molecular interaction between ERK5 and Nrf2 was further induced by laminar flow. Finally, flow-dependent nuclear localization of Nrf2 was inhibited by BIX02189, a specific inhibitor of MEK5, in aorta of mice in vivo. Collectively, these data demonstrate that laminar flow-induced activation of ERK5-Nrf2 signal pathway plays a critical role for anti-inflammatory and anti-apoptotic mechanism in endothelial cells.