Updated project metadata. Background & Aims Barrett’s esophagus (BE), a metaplastic transformation driven by gastroesophageal reflux disease (GERD), induces oxidative stress but the underlying redox mechanisms remain poorly understood. Protein persulfidation (PSSH), a redox-sensitive, reversible, and antioxidative post-translational modification regulated by hydrogen sulfide (H₂S) metabolism, has not been explored in this context. Here, we identify epithelial PSSH as a key regulator of this premalignant process. Methods We applied proteomics and chemoproteomics in patients to map and validate PSSH and total proteome profiles across healthy (squamous), GERD-exposed, and metaplastic epithelium. Using in vitro and in vivo models of chronic GERD and BE, we modulated H₂S levels genetically and pharmacologically. Mechanistic and functional effects were assessed using tissue biopsies or recombinant human proteins. Results GERD-induced oxidative loss of H₂S and its enhanced catabolism initiated early PSSH proteome remodeling in squamous epithelium, which expanded in BE and affected >1,300 proteins in clinical samples, indicating potential biomarkers. This also included altered persulfidation of enzymes regulating the accumulation of prostaglandin E₂ (PGE₂), a well-established driver of BE development and progression. H₂S depletion accelerated metaplastic transformation, whereas H₂S donors reversed these effects in experimental models. PSSH of 15-hydroxyprostaglandin dehydrogenase (PGDH) reversibly suppressed its activity, protecting the enzyme and, unlike irreversible oxidation, allowing recovery of PGE₂ degradation. Conclusions These findings redefine the origin of PGE₂ accumulation in metaplasia and establish sulfide loss and persulfidomic remodeling as central, druggable drivers of epithelial reprogramming and redox imbalance in BE pathogenesis.