Idiopathic pulmonary fibrosis (IPF) is a highly fatal disease that lacks appropriate treatments and highly effective drugs. Many reported indicated that the TGF-β1/Smad3 signaling pathway played a pivotal role in development of IPF. In this case, it was hypothesized that discovery novel compounds to block the TGF-β1/Smad3 signaling pathway might be useful for treatment of IPF. Therefore, a high-throughput screening system based on stably transfected CAGA-NIH3T3 cells was established for discovering lead compounds which could validly suppress the TGF-β1/Smad3 signal path. In this study, a series of novel Pleuromutilin derivatives were prepared and quickly evaluated by high-throughput assay. The lead compound 32 was discovered to be able to remarkably suppress the TGF-β1/Smad3 pathway in vitro. Further biological evaluation revealed that compound 32 could remarkably decrease the myofibroblast stimulation and extracellular matrix (ECM) deposition. More importantly, compound 32 could remarkably mitigate bleomycin (BLM)-triggered lung fibrosis in mice models. Additionally, the lead compound possess excellent pharmacokinetics properties, good oral availability and low toxicity. In general, our study has demonstrated the potency of a novel Pleuromutilin derivative (compound 32), which might be a prospective candidate for developing anti-IPF medicines by suppress the TGF-β1/Smad3 signal pathway.