Transforming growth factor (TGF)-β1 has long been regarded as a central mediator of tissue fibrosis. Follistatin-like 1 (Fstl1) is a crucial pro-fibrotic glycoprotein that is up-regulated in fibrotic lung tissues and that promotes fibrogenesis via facilitating TGF-β signaling. Here we examined the signaling pathway by which TGF-β1 up-regulates Fstl1 expression in mouse pulmonary fibroblasts. TGF-β1 regulated Fstl1 expression at both the transcriptional as well as translational levels. Transforming growth factor (TGF)-β1 has long been regarded as a central mediator of tissue fibrosis. Follistatin-like 1 (Fstl1) is a crucial pro-fibrotic glycoprotein that is up-regulated in fibrotic lung tissues and it promotes fibrogenesis via facilitating TGF-β signaling. Here we examined the signaling pathway by which TGF-β1 up-regulates Fstl1 expression in mouse pulmonary fibroblasts. TGF-β1 regulated Fstl1 expression at both the transcriptional and translational levels. Although TGF-β1 rapidly activated the Smad, MAPK, and Akt pathways in lung fibroblasts, only Smad2/3 inhibition eliminated TGF-β1-induced Fstl1 expression. Analysis of the luciferase reporter activity identified a functional c-Jun transcription site in the Fstl1 promoter. Our results suggested a critical role for the Smad3-c-Jun pathway in the regulation of Fstl1 expression by TGF-β1 during fibrogenesis.