Sumoylation has emerged as an indispensable post‐translational modification that modulates the functions of a broad spectrum of proteins. Recent studies have demonstrated that reactive oxygen species influence the equilibrium of sumoylation‐desumoylation. We show herein that H₂O₂ induces formation of an intermolecular disulfide linkage of human SUMO protease SENP1 via the active‐site Cys 603 and a unique residue Cys 613. Such reversible modification confers a higher recovery of enzyme activity, which is also observed in yeast Ulp1, but not in human SENP2, suggesting its protective role against irreversible sulfhydryl oxidation. In vivo formation of a disulfide‐linked dimer of SENP1 is also detected in cultured cells in response to oxidative stress. The modifications are further elucidated by the crystal structures of Ulp1 with the catalytic cysteine oxidized to sulfenic, sulfinic, and sulfonic acids. Our findings suggest that, in addition to SUMO conjugating enzymes, SUMO proteases may act as redox sensors and effectors modulating the des‐umoylation pathway and specific cellular responses to oxidative stress.— Xu, Z., Lam, L. S. M., Lam, L. H., Chau, S. F., Ng, T. B., Au, S. W. N. Molecular basis of the redox regulation of SUMO proteases: a protective mechanism of intermolecular disulfide linkage against irreversible sulfhydryl oxidation. FASEB J. 22, 127–137 (2008)