BACKGROUND: To investigate the beneficial effect of berberine on gastroesophageal reflux-induced airway hyperresponsiveness (GERAHR) and explore the underlying mechanism. METHODS: Coword cluster analysis and strategic coordinates were used to identify hotspots for GERAHR research, and an online tool (STRING, https://string-db.org/) was used to predict the potential relationships between proteins. Guinea pigs with chemically induced GERAHR received PBS or different berberine-based treatments to evaluate the therapeutic effect of berberine and characterize the underlying mechanism. Airway responsiveness was assessed using a plethysmography system, and protein expression was evaluated by western blotting, immunohistochemical staining, and quantitative PCR analysis. RESULTS: Bioinformatics analyses revealed that TRP channels are hotspots of GERAHR research, and TRPA1 is related to the proinflammatory neuropeptide substance P (SP). Berberine, especially at the middle dose tested (MB, 150 mg/kg), significantly improved lung function, suppressed inflammatory cell infiltration, and protected inflammation-driven tissue damage in the lung, trachea, esophagus, and nerve tissues in GERAHR guinea pigs. MB reduced the expression of TRPA1, SP, and tumor necrosis factor-alpha (TNF-) in evaluated organs and tissues. Meanwhile, the MB-mediated protective effects were attenuated by simultaneous TRPA1 activation. CONCLUSIONS: Mechanistically, berberine was found to suppress GERAHR-induced upregulation of TRPA1, SP, and TNF- in many tissues. Our study has highlighted the potential therapeutic value of berberine for the treatment of GERAHR.