BackgroundSilica-induced pulmonary fibrosis (silicosis) is a diffuse interstitial fibrotic disease characterized by the massive deposition of extracellular matrix in lung tissue. Fibroblast to myofibroblast differentiation is crucial for the disease progression. Inhibiting myofibroblast differentiation may be an effective way for pulmonary fibrosis treatment.MethodsThe experiments were conducted in TGF-β treated human lung fibroblasts to induce myofibroblast differentiation in vitro and silica treated mice to induce pulmonary fibrosis in vivo.ResultsBy quantitative mass spectrometry, we revealed that proteins involved in mitochondrial folate metabolism were specifically upregulated during myofibroblast differentiation following TGF-β stimulation. The expression level of proteins in mitochondrial folate pathway, MTHFD2 and SLC25A32, negatively regulated myofibroblast differentiation. Moreover, plasma folate concentration was significantly reduced in patients and mice with silicosis. Folate supplementation elevated the expression of MTHFD2 and SLC25A32, alleviated oxidative stress and effectively suppressed myofibroblast differentiation and silica-induced pulmonary fibrosis in mice.ConclusionOur study suggests that mitochondrial folate pathway regulates myofibroblast differentiation and could serve as a potential target for ameliorating silica-induced pulmonary fibrosis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-023-04241-0.