In this study, RNA interference technique was employed to silence the expression of DNMT1 and/or DNMT3b in human bladder cancer T24 cells. The expression levels of their mRNA and protein were greatly decreased by up to 75% and 65% respectively after T24 cells were transfected with lipofectamine2000 for 72 h, indicating RNA interference is an effective tool in gene knockdown. Proliferation and apoptosis of T24 cells were detected by MTT, and annexin-V-FITC and propidium iodide staining flow cytometry, respectively. It was found that loss of the DNMT1 or DNMT3b expression could inhibit the cell growth and promote the cell apoptosis to some extent. However, combined treatment with shRNA targeting both DNMT1 and DNMT3b mRNA could obviously enhance the above effects. It was concluded that simultaneously silencing both genes could result in strong suppressing effect on tumor proliferation and promoting cell apoptosis than separate use, suggesting combined use of DNMT1 and DNMT3b can achieve a synergistic effect in the CpG island methylation in human bladder tumorigenesis.