Altered hemodynamics contribute to the pathophysiology of human varicose veins. However, the molecular mechanisms connecting altered venous flow to endothelial dysfunction remain unknown. This study explored NRF2 antioxidant defense signaling in varicose veins and its regulation by distinct shear stress patterns of venous blood flow. Comparative analysis of saphenous veins from 25 patients with varicose veins and 20 control subjects using qRT-PCR, western blots and immunohistochemistry revealed a significant downregulation of NRF2 and its downstream antioxidant genes such as NQO1, SQSTM1, TXNRD1 , and GCLM in varicose veins. This was accompanied by upregulated KEAP1, which indicated an impaired NRF2-mediated cytoprotective signaling. Human umbilical vein endothelial cells (HUVECs) were exposed to physiological laminar parallel and pathological oscillatory shear stress using a microfluidic flow system to replicate the hemodynamic environment. Parallel flow exposure enhanced NRF2 and suppressed KEAP1 in HUVECs, while oscillatory flow reversed these effects. A non-canonical NRF2 repressor, WDR23, was increased by oscillatory flow and elevated in varicose veins. This indicated a dual regulation of NRF2 under altered hemodynamics. Puerarin, a natural isoflavonoid, suppressed endothelial KEAP1 and WDR23, restored NRF2 expression, and promoted its nuclear localization under ensuing oscillatory flow. Puerarin effectively reduced intracellular ROS and lipid peroxidation levels in oscillatory flow-exposed endothelial cells. This study demonstrates that oscillatory venous shear stress disrupts NRF2-mediated antioxidant defense via KEAP1 and WDR23 repression, contributing to endothelial oxidative injury. The ability of Puerarin to restore NRF2 signaling and attenuate oxidative stress suggests its potential as a mechanobiology-based therapeutic candidate for chronic venous disease.