Expression of DNA methyltransferase 1 (DNMT1), which plays an important role on aberrantly methylated CpG in the promoter regions of tumor suppressor genes (TSGs), is higher in bladder cancer cells than in normal bladder cells. Therefore, its overexpression is closely related to tumor formation. In this study, the eukaryotic vector pshRNA-DNMT1 was constructed and transfected into T24 cells. Levels of DNMT1 mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Relative to the blank control at the 24th, 48th and 72nd hour after transfection of pshRNA-DNMT1, the inhibitory rates of DNMT1 mRNA levels in T24 cells were 28.44%, 52.48%, 70.91%, respectively. Those of DNMT1 proteins were 24.27%, 57.79%, and 77.74%, respectively. Proliferation and apoptosis were assayed by MTT and flow cytometry with Annexin-V-FITC/PI staining. The growth inhibition rates of pshRNA-DNMT1 at the 24th, 48th and 72nd hour after transfection of pshRNA-DNMT1 were (4.34 ± 0.76)%, (9.87 ± 1.54)% and (13.78 ± 1.93)%, respectively. There were statistically significant differences between pshRNA-DNMT1 and the control blank at each time points (P < 0.01); 24, 48 and 72 hours after T24 cells were transfected by pshRNA-DNMT1, the apoptosis rates of pshRNA-DNMT1 were (3.87 ± 0.81)%, (8.69 ± 1.23)% and (11.46 ± 1.24)%, respectively (P < 0.01 vs blank control). Based on this case, our conclusion is that the recombinant plasmid pshRNA-DNMT1 can silence the expression of gene DNMT1 mRNA and protein effectively, and to some extent, it also can inhibit the proliferation of bladder cancer cell and promote the cellular apoptosis.