--- - |2+ Abstract Aims Clinical trials have shown the beneficial effects of exercise training against pulmonary fibrosis. This study aimed to investigate whether prophylactic intervention with exercise training attenuates lung fibrosis via modulating endogenous hydrogen sulphde (H2S) generation. Methods First, ICR mice were allocated to Control, Bleomycin, Exercise, and Bleomycin + Exercise groups. Treadmill exercise began on day 1 and continued for 4 weeks. A single intratracheal dose of bleomycin (3 mg/kg) was administered on day 15. Second, ICR mice were allocated to Control, Bleomycin, H2S, and Bleomycin + H2S groups. H2S donor NaHS (28 μmol/kg) was intraperitoneally injected once daily for 2 weeks. Results Bleomycin‐treated mice exhibited increased levels of collagen deposition, hydroxyproline, collagen I, transforming growth factor (TGF)‐β1, Smad2/Smad3/low‐density lipoprotein receptor‐related proteins (LRP‐6)/glycogen synthase kinase‐3β (GSK‐3β) phosphorylation, and Smad4/β‐catenin expression in lung tissues (P < 0.01), which was alleviated by exercise training (P < 0.01 except for Smad4 and phosphorylated GSK‐3β: P < 0.05). Bleomycin‐induced lung fibrosis was associated with increased α smooth muscle actin (α‐SMA) and decreased E‐cadherin expression (P < 0.01). Double immunofluorescence staining showed the co‐localization of E‐cadherin/α‐SMA, indicating epithelial‐mesenchymal transition (EMT) formation, which was ameliorated by exercise training. Moreover, exercise training restored bleomycin‐induced downregulation of cystathionine‐β‐synthase (CBS) and cystathionine‐γ‐lyase (CSE) expression, as well as H2S generation in lung tissue (P < 0.01). NaHS treatment attenuated bleomycin‐induced TGF‐β1 production, activation of LRP‐6/β‐catenin signalling, EMT and lung fibrosis (P < 0.01 except for β‐catenin: P < 0.05). Conclusion Exercise training restores bleomycin‐induced downregulation of pulmonary CBS/CSE expression, thus contributing to the increased H2S generation and suppression of TGF‐β1/Smad and LRP‐6/β‐catenin signalling pathways, EMT and lung fibrosis. - 'Acta Physiologica, EarlyView. '