The present study aimed to investigate the mechanism of anti-proliferative, anti-inflammatory and anti-fibrotic effects of triptolide (TPL) on activated lung fibroblasts by regulating the focal adhesion kinase (FAK) and calpain signaling pathways. The HFL-1 human foetal lung fibroblast cell line was cultured in vitro and treated with 50 ng/ml transforming growth factor (TGF)-β1 for 48 h to establish the model of pulmonary fibrosis. Subsequently, the cells were divided into five groups, including a control, model, TPL, FAK inhibitor and calpeptin group. Subsequently, the proliferation of lung fibroblasts was detected using the Cell Counting Kit-8 assay. The concentration of interleukin (IL)-6 in the cell culture supernatant was examined by ELISA and the mRNA expression levels of collagen type I (ColI)α and ColIII in lung fibroblasts were quantified by reverse transcription-quantitative PCR. The protein levels of FAK, phosphorylated (p)-FAK, calpain 1 and calpain 2 were detected by western blot analysis. TGF-β1 induced the proliferation of lung fibroblasts, whereas TPL inhibited this proliferation in a dose-dependent manner. TPL also decreased the TGF-β1-induced production of IL-6 and reduced the upregulation of ColIα, ColIII, FAK, p-FAK, and inhibited the decrease of calpain 1 and calpain 2 induced by TGF-β1. In addition, the FAK inhibitor acted synergistically with TPL to decrease TGF-β1-induced production of IL-6 and attenuate TGF-β1-induced synthesis of ColIα and ColIII, while calpeptin had an antagonistic effect on the function of TPL. Furthermore, treatment with the FAK inhibitor and TPL markedly decreased the protein levels of FAK and p-FAK, and increased the protein expression of calpain 1 and calpain 2 in lung fibroblasts stimulated by TGF-β1 to a greater extent than TPL alone, while calpeptin had an antagonistic effect on the action of TPL. In conclusion, the present study indicated that TPL protected against TGF-β1-induced proliferation, inflammation and fibrosis by regulating the FAK and calpain signaling pathways. Copyright: © Zhang et al.