Alantolactone (Ala) has a variety of biochemical properties including anti-cancer, anti-inflammation, anti-microbial and anti-fungal. However, whether Ala can attenuate renal fibrosis remains unknown. We investigated whether Ala can attenuate renal fibrosis and further defined the effect of Ala on transforming growth factor (TGF)-signaling pathways in human tubular epithelium-like cell (HK-2) and rat kidney fibroblast (NRK-49F). In the mouse unilateral ureteral obstruction (UUO) model, Ala significantly improved UUO-induced renal tubulointerstitial damage and fibrosis as evidenced by H&E, Sirius red and Masson’s trichrome staining. Ala in cultured HK-2 inhibited TGF-β-stimulated expression of collagen type I and PAI-1, but recovered TGF-β-induced decrease in E-cadherin expression. In NRK-49F, Ala inhibited TGF-β-stimulated expression of fibronectin, collagen type I, PAI-1 and α-smooth muscle actin (α-SMA). Ala inhibited TGF-β-stimulated PAI-1 promoter activity as well as the phosphorylation of Smad3 in HK-2 and NRK-49F. Our result suggest that Ala ameliorates renal fibrosis through inhibition of TGF-β/Smad3 signaling pathway and is a possible therapeutic reagent for chronic kidney disease.