Abstract: Objective To investigate the P16 gene methylation state in human colorectal cancer cell line Caco-2 and explore the possibility of re-expression of the hypermethylated and silenced P16 gene. Methods cell line Caco-2 treated with different concent rations of DNA methyltransferase inhibitor 5-Aza-CdR, MSP was used to detect promoter methylation state of P16 gene and RT-PCR were used to detect re-expression of mRNA before and after treatment with 5-Aza-CdR respectively. Cell growth speed was measured using MTT assay, cell cycle distribution and apoptosis rate were estimated using ?ow cytometry. Results Promoter hypermethylation of the P16 gene was detected in human colorectal cancer cell line Caco-2, After treatment with 5-Aza-CdR, the promoter region of the P16 gene exhibited a demethylation state, and 5-Aza-CdR induces P16 gene re-expression. 5-Aza-CdR effectively reversed the hypermethylation status of CpG island,induced colorectal cancer cell apoptosis and cell cycle arrest were observed in a dose -dependent manner. Conclusion P16 gene can be reversed by demethylation agent 5-Aza-CdR. demethylation agent can regulate the expression of the P16 gene,and effectively inhibits cell growth.