Abstract: Objective To investigate the relationship between expression of tumor suppressor gene p16 in non-small cell lung cancer (NSCLC) tissues and clinicopathological parameters, to further study on DNA methyltransferase inhibitors 5-nitrogen impurity-2′-deoxycytidine (5-Aza-CdR) in human lung cancer cell line A549 in regulating the expression of p16. Methods The expression of p16 protein in 76 cases of NSCLC tissues and normal tissue adjacent to carcinoma were detect by immunohistochemical SP method and the differences of p16 protein expression were analyzed. p16 gene promoter region of DNA methylation status were detect by MSP method in 5-Aza-CdR processing A549 cells, the expression of p16 in A549 lung cancer cell and effect of 5-Aza-CdR were detect by Western blot method. Results 32 cases (42.11%) of p16 protein expression was positive, significantly lower than that of the normal tissue adjacent to carcinoma (positive expression in 59 cases, 77.63%) in 76 cases of NSCLC tissues; There were statistically significant differences (P<0.05) in the positive expression rates of p16 in NSCLC tissues with different pathological tissue grading, tumor differentiation degree, clinical TNM stage and lymph node metastasis. In A549 cells, p16 protein expression and non-methylated products were both in low expression states. After treated with 5-Aza-CdR, the expression of p16 protein and its non-methylated products were up-regulated, with the increase of 5-aza-cdr concentration. Conclusion The low expression of p16 in NSCLC tissues with squamous cell carcinomas, low differentiation, lymph node metastasis and phase Ⅲ-Ⅳ, which may prompt the deactivation and cause further progression of NSCLC, 5-Aza-CdR could induce the expression of p16 protein and non-methylated products in A549 cells.