Fluid shear stress has been revealed to differentially regulate endothelial nitric oxide synthase (eNOS) distribution in vessels. eNOS, a key enzyme in controlling nitric oxide (NO) release, has a crucial role in mediating oxidative stress, and resveratrol (RSV)‑mediated eNOS also attenuates oxidative damage and suppresses endothelial dysfunction. To observe the protective effect of RSV on low shear stress (LSS)‑induced oxidative damage and the potential mechanisms involved, a parallel‑plate flow chamber, which imposed a low level of stress of 2 dynes/cm2 to cells, was employed. Reactive oxygen species (ROS), NO and apoptotic cells were examined in LSS‑treated endothelial cells (ECs) with or without RSV. Western blot analysis was used to examine LSS‑regulated eNOS‑Ser1177, Thr495 and Ser633, which were tightly associated with NO release. To further determine the underlying signaling pathways involved, extracellular signal‑regulated kinase (ERK), a possible upstream target of eNOS‑Thr495, was investigated, followed by examination of eNOS‑Thr495 in ERK‑inhibited cells. Additionally, eNOS mRNA expression levels were analyzed in cells challenged with LSS. The results revealed that RSV markedly decreased LSS‑induced oxidative damage in ECs. Furthermore, eNOS‑Ser1177 and Thr495 as well as phospho‑ERK were time‑dependently activated by LSS. The ERK inhibitor deactivated eNOS‑Thr495, which was accompanied by increased intracellular superoxide dismutase (SOD) levels. Of note, the activation effect of LSS on ERK/eNOS was markedly eliminated by RSV. In conclusion, RSV exerts antioxidant effects by suppressing LSS-activated ERK/eNOS and may provide a potential therapeutic target for atherosclerosis.