Hydroxysafflor yellow A (HSYA) is an active ingredient of Carthamus tinctorius L.. This study aimed to evaluate the effects of HSYA on transforming growth factor-beta 1 (TGF-beta-1)-induced changes in proliferation, migration, differentiation, and extracellular matrix accumulation and degradation in human fetal lung fibroblasts (MRC-5), to explore the mechanisms whereby HSYA may alleviate pulmonary fibrosis. MRC-5 cells were incubated with various doses of HSYA and/or the TGF-beta receptor type I kinase inhibitor SB431542 and then stimulated with TGF-beta 1. Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium inner salt assay. Cell migration was detected by wound-healing assay. Protein levels of alpha-smooth muscle actin (alpha-SMA), collagen I alpha 1 (COL1A1), and fibronectin (FN) were measured by immunofluorescence. Protein levels of matrix metalloproteinase-2, tissue inhibitor of matrix metalloproteinase-1, tissue inhibitor of matrix metalloproteinase-2, TGF-beta type II receptor (T beta RII), and TGF-beta type I receptor were detected by western blotting. T beta RII knockdown with siRNA interfered with the inhibitory effect of HSYA on alpha-SMA, COL1A1, and FN expression, and TGF-beta-1-induced Sma and Mad protein (Smad), and extracellular signal-regulated kinase/mitogenactivated protein kinase signaling pathway activation. The antagonistic effect of HSYA on the binding of fluorescein isothiocyanate-TGF-beta-1 to MRC-5 cell cytoplasmic receptors was measured by flow cytometry. HSYA significantly suppressed TGF-beta-1-induced cell proliferation and migration. HSYA could antagonize the binding of FITC-TGF-beta-1 to MRC5 cell cytoplasmic receptors. Also HSYA inhibited TGF-beta-1-activated cell expression of a-SMA, COL1A1, and FN and phosphorylation level of Smad2, Smad3, and ERK by targeting T beta RII in MRC-5 cells. These findings suggest that T beta RII might be the target responsible for the inhibitory effects of HSYA on TGF-beta-1-induced pathological changes in pulmonary fibrosis.