Oxidative maturation of secretory and membrane proteins in the endoplasmic reticulum (ER) is powered by Ero1 oxidases. To prevent cellular hyperoxidation, Ero1 activity can be regulated by intramolecular disulphide switches. Here, we determine the redox-driven shutdown mechanism of Ero1, the housekeeping Ero1 enzyme in human cells. We show that functional silencing of Ero1 in cells arises from the formation of a disulphide bond-identified by mass spectrometry-between the active-site Cys⁹⁴ (connected to Cys⁹⁹ in the active enzyme) and Cys¹³¹. Competition between substrate thiols and Cys¹³¹ creates a feedback loop where activation of Ero1 is linked to the availability of its substrate, reduced protein disulphide isomerase (PDI). Overexpression of Ero1-Cys131Ala or the isoform Ero1, which does not have an equivalent disulphide switch, leads to augmented ER oxidation. These data reveal a novel regulatory feedback system where PDI emerges as a central regulator of ER redox homoeostasis.