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Background: This study was conducted to investigate the effect of P14 promoter aberrant methylation on the biological function of human lung adenocarcinoma cells. Methods: We used nested methylation‐specific PCR (NMSP) to detect the methylation status of the p14ARF promoter region in SPCA1 and BEAS2B cell lines. The experimental groups were treated with 5‐aza‐2′‐deoxycytidine (5‐Aza). Quantitative real‐time PCR, Western blot, flow cytometry, and Cell Counting Kit 8 were used to detect the expression of p14ARF messenger RNA and protein in each group, apoptosis, and cell proliferation inhibition, respectively. Results: NMSP detected that the p14 promoter region of SPCA1 cells has abnormal methylation status. After treatment with 5‐Aza, the expression of p14ARF messenger RNA and protein in SPCA1 cells (P < 0.05) and the inhibition rate of cell proliferation (P < 0.05) were significantly increased, while the apoptosis rate was markedly increased (P < 0.05). However, no differences were observed in BEAS2B cells (P > 0.05). Conclusion: Abnormal methylation of the p14ARF promoter region plays an important role in the development of lung cancer cells. Our results suggest the use of P14 promoter aberrant methylation as a therapeutic target for drug research or to improve the sensitivity of other drugs. [ABSTRACT FROM AUTHOR]