Pulmonary fibrosis is the end stage of many interstitial lung diseases, characterized by the deposition of excess extracellular matrix (ECM), destruction of normal alveolar structure, and resulting in the obstruction of gas exchange and respiratory failure. The idiopathic pulmonary fibrosis (IPF) is the most common form of pulmonary fibrosis with little effective therapies. 5-Methoxytryptophan (5-MTP) is a newly found tryptophan metabolite. Previous studies suggested that 5-MTP has the effects of anti-inflammatory, anti-tumorigenesis, vascular protection and anti-fibrosis in renal disease. Whether 5-MTP has therapeutic effect on pulmonary fibrosis is not clear. In our study, we used TGF-β1 to stimulate human lung fibroblasts (HLFs) and bleomycin (BLM) induced pulmonary fibrosis model to investigate the effect of 5-MTP on pulmonary fibrosis. Our study demonstrated that 5-MTP could improve the lung function and attenuate the destruction of alveolar structure in BLM-induced pulmonary fibrosis mice. Furthermore, 5-MTP significantly decreased accumulation of myofibroblasts and the deposition of ECM by inhibiting the differentiation of fibroblasts to myofibroblasts and suppressing the protein expression of the ECM both in vivo and in vitro. Our results also revealed 5-MTP could inhibit the proliferation and migration of the fibroblasts in vitro, which played an important role in the progressive pulmonary fibrosis. To further investigate the mechanism of the anti-fibrosis of 5-MTP, several canonical and noncanonical signaling pathways were examined. Our results revealed that 5-MTP could inhibit the pulmonary fibrosis through downregulating the phosphorylation of TGF-β/SMAD3, PI3K/AKT signaling pathways. Together, our study indicated that 5-MTP promises to be therapeutic agent of pulmonary fibrosis.Copyright © 2018. Published by Elsevier Inc.