Macroautophagy/autophagy plays a pivotal role in cytoplasmic material recycling and metabolic turnover, in which ATG4B functions as a "scissor" for processing pro-LC3 and lipidated LC3 to drive the autophagy progress. Mounting evidence has demonstrated the tight connection between ROS and autophagy during various pathological situations. Coincidentally, several studies have shown that ATG4B is potentially regulated by redox modification, but the underlying molecular mechanism and its relationship with autophagy is ambiguous. In this study, we verified that ATG4B activity was definitely regulated in a reversible redox manner. We also determined that Cys292 and Cys361 are essential sites of ATG4B to form reversible intramolecular disulfide bonds that respond to oxidative stress. Interestingly, we unraveled a new phenomenon that ATG4B concurrently formed disulfide-linked oligomers at Cys292 and Cys361, and that both sites underwent redox modifications thereby modulating ATG4B activity. Finally, increased autophagic flux and decreased oxidation sensitivity were observed in Cys292 and Cys361 double site-mutated cells under normal growth conditions. In conclusion, our research reveals a novel molecular mechanism that oxidative modification at Cys292 and Cys361 sites regulates ATG4B function, which modulates autophagy.Abbreviations: Air-ox: air-oxidation; ATG4B: autophagy related 4B cysteine peptidase; BCNU: 1,3-bis(2-chloroethyl)-1-nitrosourea; CBB: Coomassie Brilliant Blue; CM: complete medium; CQ: chloroquine; DTT: dithiothreitol; GSH: reduced glutathione; GSNO: S-nitrosoglutathione; GSSG: oxidized glutathione; HMW: high molecular weight; H2O2: hydrogen peroxide; NAC: N-acetyl-L-cysteine; NEM: N-ethylmaleimide; PE: phosphatidylethanolamine; PTM: post-translational modification; ROS, reactive oxygen species; WT: wild type.