We have determined the impact of the oxidoreductase chaperone protein disulfide isomerase (PDI) on the critical structure-forming step during the oxidative maturation of model disulfide-bond-containing proteins. This is achieved by using a novel tool to trap and populate native-disulfide-containing intermediates in unstructured forms that are poised to fold. Our data reveals that PDI inhibits the conformational folding step of oxidative fold maturation and, therefore, has limited overall catalytic efficiency as an oxidoreductase chaperone. Such an anomalous behavior of PDI during a key step in oxidative regeneration may contribute to misfolding in the endoplasmic reticulum, aggregation, and neurodegenerative disease.