Idiopathic pulmonary fibrosis (IPF) is an interstitial fibrotic lung disease characterized by myofibroblast differentiation and collagen deposition. Excessive activation of fibroblasts in the lungs leads to severe alveolar dysfunction and tissue destruction seen by histological assessment. IPF presents a high mortality rate, limited therapeutic options, and an intense need to develop safe and effective therapeutic drugs. Lysionotin is a flavonoid isolated from herbal extracts with various biological effects such as anti-tuberculosis mycobacteria and anti-inflammatory. Nevertheless, its effect on pulmonary fibrosis is not known. This study aims to investigate the effect of Lysionotin on bleomycin (BLM)-induced pulmonary fibrosis and its mechanism. We used BLM to establish a mouse model of pulmonary fibrosis and injected Lysionotin intraperitoneally on days 15-28 to observe its effect on pulmonary fibrosis. The molecular mechanism of Lysionotin was investigated in vitro using transforming growth factor-β (TGF-β) induced myofibroblasts. Lysionotin attenuates TGF-β-induced myofibroblast differentiation and oxidative stress by promoting nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream expression of antioxidant genes NAD(P)H quinone dehydrogenase 1 (NQO-1) and heme oxygenase 1 (HO-1) by activating AMP-activated protein kinase (AMPK). Lysionotin exerts anti-pulmonary fibrosis effects by regulating myofibroblast differentiation and reducing oxidative stress through the AMPK/Nrf2 pathway, illustrating the potential significance of Lysionotin in protecting against BLM-induced pulmonary fibrosis.