Redox state is a critical factor determining enzyme activity. The protein disulfide isomerase (PDI) belongs to a class of enzymes that maintains proteins in the adequate redox state by isomerising thiols. Vascular NADPH oxidases are important sources of reactive oxygen species (ROS). As NADPH oxidases are likely to be thiol oxidized by the radicals generated by themselves, we hypothesize that the PDI promotes oxidative stress in vascular cells by maintaining NADPH oxidase activity. Overexpression of fluorescence-tagged fusion constructs revealed colocalization of the NADPH oxidase proteins Nox1, Nox2 and Nox4 with endogenous PDI. Moreover, immuno-precipitation of PDI resulted in co-precipitation of Nox 1, 2 and 4 as well as of the p22phox subunit of the NAPDH oxidase. ROS formation in NADPH oxidase-over-expressing HEK cells was reduced by siRNA directed against PDI. Analysis of NADPH oxidase homologues revealed several highly conserved cysteines. Site-directed mutagenesis to valine or serine lead to the identification of two essential cysteines, which maintain activity independent of steric effects. Therefore, these cysteins are likely to be redox modulated and to be targets of PDI. In vascular smooth muscle cells (VSMC) downregulation of PDI by siRNA attenuated NADPH oxidase activity and significantly impaired the agonist-induced secretion of the oxidative-stress-inducible chemokine MCP-1. Therefore, PDI is a novel regulator of NADPH oxidases.