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Background/Aims: BushenShugan Formula (BSF) is a traditional Chinese medicine that has therapeutic effects on middle- and late-stage lung adenocarcinoma in clinical application. It was reported that Bushen Chinese medicine suppressed the onset of pre-metastatic niches in a murine model of spontaneous lung metastasis. However, the mechanisms of BSF on human lung adenocarcinoma remain unknown. Methods: Cell proliferation was determined by CCK8 and colony formation. Cell apoptosis and cell cycle were detected by flow cytometry. Cancer stem cells properties were examined by spheroid body formation. The migration and invasion abilities were analyzed by wound healing assay and transwell invasion assay. The mRNA expressions were determined by qRT-PCR. Western blotting analysis showed the protein levels. Results: BSF was shown to inhibit the proliferation of A549 cells in time- and concentration-dependent manners. Colony formation assays also indicated the antiproliferative effect of BSF against A549 cells. Cellular mechanistic studies demonstrated that BSF arrested the cell cycle in G2/M phase and induced apoptosis. Importantly, BSF could inhibit the epithelial-mesenchymal transition(EMT) of A549 cells through PI3K/AKT/NF-κB pathway. Conclusions: BSF effectively inhibited tumour growth, suggesting that it is a promising anticancer treatment for further clinical development. [ABSTRACT FROM AUTHOR]