Barrett’s esophagus (BE) is a precancerous consequence of gastroesophageal reflux disease (GERD) where metaplastic epithelium replaces squamous mucosa. Mechanisms driving this transformation still remain unclear. Posttranslational proteins persulfidation emerged significant in aging and cardiovascular system, but their role in gastrointestinal (GI) disorders remains unknown. Persulfidation relies on the endogenous hydrogen sulfide (H2S) signaling. We hypothesized that pathway-specific persulfidation impairments underlie BE pathogenesis. We aimed to assess if H2S-bioavailability restoration/depletion impacts persulfidation-sensitive targets and BE development. Methods We analyzed total and persulfidated proteome in 3-sets of clinical biopsies representing BE and squamous epithelium +/- GERD. We implemented an animal model of GERD and BE, generating esophago-gastroduodenal anastomosis (EGDA), followed by 10-weeks-long treatments with H2S donors/inhibitors. We assessed gastric secretion in rats. We employed in vitro human-derived healthy (EPC2), GERD-exposed (NES-G2T) and Barrett’s (BAR-T) cells +/- CRISPR-Cas9-induced H2S-producing enzymes knockouts. Results GERD-induced clinical persulfidation changes in squamous epithelium expanded in BE. Persulfidations of interleukin (IL)-1, prostaglandin (PG)E2 and mTOR signaling, but not exclusively, were impaired. GERD decreased H2S bioavailability in rats esophagus and human squamous, but not metaplastic cells. H2S, reconstituted by i.g. treatment with NaHS or GYY4137, inhibited gastric secretion, metaplasia development, nucleic acids oxidation, IL-1/PGE2/mTOR activation, restoring mucosal microcirculation. H2S-enzymes inactivation accelerated morphological and molecular turnover. Total proteome clustering indicated, among others, NAD(P)H quinone dehydrogenase 1 as an early BE biomarker. GERD-altered persulfidation in squamous epithelium initiate Barrett’s metaplasia, as the mechanistic outcome of reduced H2S bioavailability. Stable H2S biosynthesis and enriched persulfidation switch reflect the adaptative GERD-resistance of the metaplastic epithelium. H2S counteracts BE development by mechanistically interfering with metaplasia-activating IL-1β/PGE2/mTOR pathways.