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Objective. This study aims to investigate the role of NAC1/HMGB1 signaling pathway in the epithelial-mesenchymal transition (EMT), invasion, and metastasis of lung cancer cell line. Methods. Human lung cancer cell line A549 was used in this study. They were randomly divided into normal control group, sh-NAC1 empty vector group (sh-NAC1 NC), expression empty vector group (NAC1 NC), NAC1-shRNA and NAC1 over-expression group (NAC1). NAC1 and HMGB1 expression levels were detected by qRT-PCR method. Cell proliferation was detected by CCK8 kit. Cell cycles were detected by flow cytometry method. Cell invasion was detected by Transwell method. The expression levels of E-cadherin, N-cadherin, NAC1, and HMGB1 were detected by qRT-PCR and Western blotting methods. Results. Compared with the control group, the expression level of NAC1 and cell proliferation in NAC1-shRNA group decreased, cells in G1 phase increased and cells in S phase decreased. In NAC1-shRNA group, E-cadherin expression levels increased and the expression levels of N-cadherin, HMGB1 and Vimentin decreased. In NAC1 group, the expression level of NAC1 and cell proliferation increased, cells in S phase increased and cells in G1 phase decreased, E-cadherin expression levels decreased and the expression levels of N-cadherin, HMGB1 and Vimentin increased. All these differences are statistically significant. Conclusions. The expression of NAC1 and HMGB1 in lung cancer cells may affect the occurrence of EMT, the NAC1/HMGB1 signaling pathway is associated with the EMT, invasion, and metastasis of lung cancer cells.