Abstract: In this study, we investigated the effect and mechanism of glaucocalyxin A on transforming growth factor-β1 (TGF-β1)-induced differentiation of lung fibroblasts by Western blotting, cellular immunofluorescence and collagen gel contraction assays. We monitored the phosphorylation of Smad3, measured extracellular regulated protein kinases (ERK) 1/2 and glycogen synthase kinse3β (Ser9) activity and the level of β-catenin to elucidate the role of glaucocalyxin A. The results show that glaucocalyxin A significantly decreased the expression of α-smooth muscle actin in lung fibroblasts; glaucocalyxin A remarkably reduced the formation of filaments and collagen gel contraction of lung fibroblasts; glaucocalyxin A notably down-regulated the production of fibronectin; glaucocalyxin A did not affect the phosphorylation level of Smad3 and ERK1/2; glaucocalyxin A markedly inhibited the phosphorylation of GSK3β (Ser9) and the levels of β-catenin; a GSK3β (S9A) mutant significantly inhibited lung fibroblast differentiation; and SKL2001, a β-catenin activator, partly reversed the inhibition of lung fibroblast differentiation by glaucocalyxin A. These results suggest that glaucocalyxin A significantly inhibits the differentiation of lung fibroblasts, which is related to the down-regulation of GSK3β/β-catenin signaling.