Fluid shear stress is intimately linked with vascular oxidative stress and atherosclerosis. We posited that atherogenic oscillatory shear stress (OSS) induced mitochondrial superoxide (mtO₂^•−) production via NADPH oxidase and c-Jun NH₂-terminal kinase (JNK-1 and JNK-2) signaling. In bovine aortic endothelial cells, OSS (±3 dyn/cm²) induced JNK activation, which peaked at 1 h, accompanied by an increase in fluorescein isothiocyanate-conjugated JNK fluorescent and MitoSOX Red (specific for mtO₂^•− production) intensities. Pretreatment with apocynin (NADPH oxidase inhibitor) or N-acetyl cysteine (antioxidant) significantly attenuated OSS-induced JNK activation. Apocynin further reduced OSS-mediated dihydroethidium and MitoSOX Red intensities specific for cytosolic O₂^•− and mtO₂^•− production, respectively. As a corollary, transfecting bovine aortic endothelial cells with JNK siRNA (siJNK) and pretreating with SP600125 (JNK inhibitor) significantly attenuated OSS-mediated mtO₂^•− production. Immunohistochemistry on explants of human coronary arteries further revealed prominent phosphorylated JNK staining in OSS-exposed regions. These findings indicate that OSS induces mtO₂^•− production via NADPH oxidase and JNK activation relevant for vascular oxidative stress. Antioxid. Redox Signal. 15, 1379–1388.