Aristolochic acid nephropathy (AAN) has become a worldwide disease and is the most severe complication related to the use of traditional Chinese medicine. However, the pathogenic mechanisms of AAN remain unclear and therapies are limited. The present study tested the hypothesis that transforming growth factor (TGF)-beta/Smad3 may be a key pathway leading to chronic AAN. This was examined in vivo in Smad3 wild-type/knockout (WT/KO) mice and in vitro in tubular epithelial cells with knockdown of Smad2 or Smad3. Results revealed that chronic administration of aristolochic acid (AA) resulted in a severe AAN characterized by progressive renal dysfunction and tubulointerstitial fibrosis including epithelial-mesenchymal transition (EMT) in Smad3 WT mice, but not in Smad3 KO mice, suggesting a critical role for Smad3 in the development of AAN. This was further tested in vitro. We found that AA was able to activate Smad signaling to mediate EMT and renal fibrosis via both TGF-beta-dependent and JNK/MAP kinase-dependent mechanisms because blockade of JNK and specific knockdown of Smad3, but not Smad2, were able to attenuate AA-stimulated collagen matrix expression and EMT. In conclusion, TGF-beta/Smad3 may be an essential mediator for chronic AAN. Results from this study indicate that specific blockade of the TGF-beta/Smad3 signaling pathway may have therapeutic potential for chronic AAN.