Chronic alterations in blood flow elicit an adaptive response that tends to normalize shear stress, involving nitric oxide (NO) and matrix metalloproteinases (MMPs). To evaluate the role of NADPH oxidase in this process, we developed a new model of mouse arteriovenous fistula (AVF) connecting the right common carotid artery (RCCA) with the jugular vein, which does not affect blood pressure. Mice deficient for gp91phox and p47phox subunits of NADPH and wild-type controls were used. AVF greatly increased RCCA blood flow (0.78±0.12 to 4.71±0.78 mL/min; P<0.01), producing an abrupt rise in shear stress (35±1 to 261±17 dynes/cm²; P<0.01) within 24 hours. RCCA diameter (460±14 μm) gradually enlarged 1 and 3 weeks after AVF (534±14 μm and 627±19 μm; P<0.01), reducing shear stress (173±13 and 106±10 dynes/cm², respectively). In gp91phox^−/− mice, changes in RCCA caliber and shear stress matched controls. However, p47phox^−/− mouse RCCAs enlarged only marginally, such that shear stress remained high (199±8 dynes/cm² at 3 weeks). Likewise, remodeling was minimal in endothelial NO synthase (eNOS)^−/− mice. In both control and gp91phox^−/− animals, reactive oxygen species (ROS) production and MMP induction was enhanced by AVF, whereas in p47phox^−/− and eNOS^−/− mice such response was negligible. Similarly, nitrotyrosine staining, indicating peroxynitrite formation, was more pronounced in control and gp91phox^−/− mice than in p47phox^−/− and eNOS^−/− mice. Hence, shear stress induces vascular NADPH oxidase comprising p47phox but not gp91phox. Generated ROS interact with NO to produce peroxynitrite, which in turn activates MMPs, facilitating vessel remodeling. Our study provides the first evidence that ROS play a fundamental role in flow-induced vascular enlargement.