Objective Hydroxysafflor yellow A (HSYA) is one of the chemical component isolated from Chinese medicine Carthamus tinctorius L. Our preliminary study confirmed that HSYA attenuated bleomycin-induced pulmonary fibrosis in mice. In this study, we evaluated the effect of HSYA on TGF-beta 1-induced activation of human fetal lung fibroblasts (MRC-5) and explored the underlying mechanisms of its activity. Method MRC-5 cells activated by TGF-beta 1 were incubated with HSYA and/or the TGF-beta type I receptor inhibitor, SB431542. TGF-beta 1-induced cell proliferation, a-smooth muscle actin, collagen I alpha 1 and fibronectin expression, Smad, mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase/Akt signalling pathway activation were observed. Key findings Hydroxysafflor yellow A significantly inhibited TGF-beta 1-induced cell proliferation and the expression, both mRNA and protein, of a-smooth muscle actin, collagen I alpha 1 and fibronectin. HSYA also suppressed TGF-beta 1 activation of Smad signal transduction via inhibition of Smad2 and Smad3 phosphorylation, their nuclear translocation and the binding activity of Smad3 to type I collagen promoter in MRC-5 cells. In addition, HSYA inhibited TGF-beta 1-induced phosphorylation of extracellular signal-regulated kinase (ERK). The inhibitory effects of HSYA were similar to SB431542. Conclusion These findings suggest that HSYA inhibits TGF-beta 1-induced activation of MRC-5 cells associated with TGF-beta 1/Smad and ERK/MAPK signalling pathways.