Epithelial-mesenchymal transition (EMT) plays important roles in the metastasis of solid tumors. In this study, the effect of toosendanin (TSN), a natural insecticide extracted from Melia toosendan Sieb et Zucc, on transforming growth factor-β1 (TGF-β1)-induced EMT was investigated. EMT was induced by TGF-β1 in A549 and H1975 lung cancer cells. The morphological alterations were observed with a microscopy. The protein expression and localization of EMT biomarkers were determined by Western blotting and immunofluorescence. The migration, invasion, and adhesion were determined by wound-healing, transwell, and adhesion assays. TGF-β1 treatment induced spindle-shaped alterations of cells, upregulation of N-cadherin, Vimentin, p-ERK1/2, and downregulation of E-cadherin. The abilities of migration, invasion, and adhesion were also enhanced. These effects were significantly reversed by TSN at very low concentration (<10 nM). Furthermore, silence Snail significantly reversed TGF-β1-induced EMT biomarkers. In addition, TGF-β1-induced phosphorylation of ERK1/2 without affecting p38 mitogen-activated protein kinases and Jun N-terminal kinase. PD98059 and U0126, inhibitors of ERK1/2, showed similar inhibitory effect to that of TSN. In summary, TSN significantly inhibited TGF-β1-induced EMT and migration, invasion, and adhesion through ERK/Snail pathway in lung cancer cells. This study provides novel anticancer effects and molecular mechanisms for TSN.