The thiol group of the amino acid cysteine can be modified to regulate protein activity. The Escherichia coli periplasm is an oxidizing environment in which most cysteine residues are involved in disulfide bonds. However, many periplasmic proteins contain single cysteine residues, which are vulnerable to oxidation to sulfenic acids and then irreversibly modified to sulfinic and sulfonic acids. We discovered that DsbG and DsbC, two thioredoxin-related proteins, control the global sulfenic acid content of the periplasm and protect single cysteine residues from oxidation. DsbG interacts with the YbiS protein and, along with DsbC, regulates oxidation of its catalytic cysteine residue. Thus, a potentially widespread mechanism controls sulfenic acid modification in the cellular environment.