Abstract: Objective To investigate the effect of DNA methylation inhibitor 5-Aza-2’-deoxycytidine (5-Aza-CdR) on the level of promoter methylation in human hepatoma cell line SMMC-7721 cells. Methods Human hepatoma cell line SMMC-7721 cells were treated with different concentrations (0.5 μmol/L, 5.0 μmol/L, 50.0 μmol/L) of 5-Aza-CdR for 72 hours, with the untreated human hepatoma cell line SMMC-7721 cells as control group. The application of pyrosequencing was used to detect the average level of promoter methylation. Results DAPK gene promoter was highly methylated in human hepatoma cell line SMMC-7721 cells, with the average methylation level as 76.71%. When the cells were treated with different concentrations（0.5 μmol/L, 5.0 μmol/L, 50.0 μmol/L）of 5-Aza-CdR for 72 hours, the average methylation level of DAPK gene for each treatment group was 78.29%, 77.57% and 66.00%, respectively, and statistically significant difference was found among different treatment groups (F = 39.71 FF ，P ＜ 0.01). The average methylation level of DAPK gene in the 50.0 μmol/L treatment group was lowest, compared with other groups (P ＜ 0.01). Conclusions 5-Aza-CdR can reverse the status of DAPK gene promoter methylation and induce the mRNA expresseion of DAPK in SMMC-7721 cells.