Objective— PDI (protein disulfide isomerase A1) was reported to support Nox1 (NADPH oxidase) activation mediated by growth factors in vascular smooth muscle cells. Our aim was to investigate the molecular mechanism by which PDI activates Nox1 and the functional implications of PDI in Nox1 activation in vascular disease. Approach and Results— Using recombinant proteins, we identified a redox interaction between PDI and the cytosolic subunit p47 ^phox in vitro. Mass spectrometry of crosslinked peptides confirmed redox-dependent disulfide bonds between cysteines of p47 ^phox and PDI and an intramolecular bond between Cys 196 and 378 in p47 ^phox . PDI catalytic Cys 400 and p47 ^phox Cys 196 were essential for the activation of Nox1 by PDI in vascular smooth muscle cells. Transfection of PDI resulted in the rapid oxidation of a redox-sensitive protein linked to p47 ^phox , whereas PDI mutant did not promote this effect. Mutation of p47 ^phox Cys 196, or the redox active cysteines of PDI, prevented Nox1 complex assembly and vascular smooth muscle cell migration. Proximity ligation assay confirmed the interaction of PDI and p47 ^phox in murine carotid arteries after wire injury. Moreover, in human atheroma plaques, a positive correlation between the expression of PDI and p47 ^phox occurred only in PDI family members with the a′ redox active site. Conclusions— PDI redox cysteines facilitate Nox1 complex assembly, thus identifying a new mechanism through which PDI regulates Nox activity in vascular disease.