Idiopathic pulmonary fibrosis (IPF) is a chronic lethal interstitial lung disease with unknown etiology. Recent studies have indicated that heat-shock protein 27 (HSP27) contributes to the pathogenesis of IPF through the regulation of epithelial-mesenchymal transition (EMT). However, the expression and role of HSP27 in fibroblasts during pulmonary fibrogenesis has not been investigated to date, at least to the best of our knowledge. In this study, we examined the expression of HSP27 in fibrotic lung tissue and fibroblasts from bleomycin (BLM)-challenged mice and human lung fibroblasts treated with transforming growth factor-beta (TGF-beta). The results revealed that the expression of HSP27 was significantly increased in fibrotic lung tissue and fibroblasts from BLM-challenged mice. In vitro, TGF-beta stimulated HSP27 expression in and the differentiation of human lung fibroblasts. The knockdown of Smad3 expression or nuclear factor-kappa B p65 subunit attenuated the TGF-beta-induced increase in HSP27 expression and the differentiation of human lung fibroblasts. In addition, the knockdown of HSP27 expression attenuated the TGF-beta-induced activation of ERK and Smad3, and inhibited the differentiation of human lung fibroblasts. On the whole, the findings of our study demonstrate that HSP27 expression is upregulated in lung fibroblasts during pulmonary fibrosis, and subsequently, HSP27 modulates lung fibroblast differentiation through the Smad3 and ERK pathways.