Objective To investigate the regulatory effect of betulin on transforming growth factor-β/Smad signaling pathway and its effect on TGF-β1-induced epithelial-mesenchymal transition process of non-small cell lung cancer. Methods The EMT model of non-small cell lung cancer A549 cells was established by exogenous TGF-β1 stimulation. The cells were divided into control group, TGF-β1 group (adding 2 μg/L TGF-β1), melitotide group (adding 2.5 mg/L melitotide), TGF-β1+ melitotide group (adding 2 μg/L TGF-β1 and 2.5 mg/L melitotide). Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the expression levels of EMT marker proteins calcadherin E, n-calcadherin, vimentin and their mRNA. Meanwhile, the protein expressions of Smad2 and p-Smad2 in each group were detected to analyze the effect of melitinin on TGF-β/ Smad signaling pathway in NSCLC. Using immunofluorescence experiments testing in the control group and TGF-βⅠ melittin group cells receptors and TGF-βⅡ receptor expression, at the same time the two groups of Transwell experiment testing cell migration, invasion ability. Results Compared with the control group, TGF-β1 group significantly decreased the expression levels of cadcadin emRNA and protein, and N-cadcadin mRNA and protein and vimentin mRNA and protein, the expression level was significantly increased ( P＜0.05 ). The expression levels of cadherin emRNA and protein in melittin + TGF-β1 group were significantly higher than those in TGF-β1 group. N-cadherin mRNA and protein in melittin+TGF-β1 group were significantly higher than those in TGF-β1 group and vimentin mRNA, protein were lower than TGF-β1 group( P＜0.05 ). In the control group and melittin group cells TGF, there was no statistically significant difference beta Ⅰ receptor expression( P＞0.05 ), TGF-beta Ⅱ receptor content in melittin group obviously less than control group. Transwell test results showed that the cell migration and invasion ability of TGF-β1 group was significantly better than that of the control group cells and The cell migration and invasion ability of betinin +TGF-β1 group was significantly worse than that of TGF-β1 group ( P＜0.05 ). Conclusion Melittin can inhibit TGF-β/SMAD signaling pathway, inhibit phosphorylation Smad2, reduce TGF-βⅡ receptors, inhibite the invasion and migration of tumor cells, and the EMT process of lung cancer cells induced by TGF-β1 was inhibited.