Abstract: Objective To construct the small interfering RNA(siRNA) expression vector targeting DNMT1 gene, and to investigate the effect of cell cycle, proliferation and apotosis of breast cancer cell line. Methods Three short hairpin RNA(shRNA) targeting coding sequence of DNMT1 were synthesized, and the cell three recombination plasmids were constructed pGCsi-DNMT1. After transfection into breast cancer MCF-7 cells, the mRNA expression level of DNMT1 gene was detected by real-time quantitative PCR, and cell cycle were analyzed by flow cytometry；The growth status of cells was detected by MTT assay, and the cell apoptosis was analyzed by Annexin V/PI double-dyed. The influence of mRNA expression about RASSF1A、p16、p21、p27 and ERβ was analyzed by real-time quantitative PCR after silencing DNMT1 gene. Results Three recombinant plasmids pGCsi-DNMT1 were successfully constructed.It was confirmed that pGCsi-T3 can markedly silence DNMT1 gene expression. Transfection of pGCsi-T3 significantly down-regulated the DNMT1 mRNA expression in MCF-7 cells. The proliferation of MCF-7 cells were markedly inhibited after transfection with pGCsi-T3, A majority of cells has become apoptosis,The frequency of S phase of cell cycle obviously reduced while G1/G0 phase significantly increased in MCF-7 cells. From the real-time PCR dectction results,it showed that the expression of RASSF1A,p16,p21 and ERβ mRNA obviously raised while the expression of p27 mRNA had no change. Conclusion PGCsi-DNMT1 can efficiently and specifically inhibit the expression of DNMT1 gene in MCF-7 cells and the cell proliferation,and promote the cell apotosis. The tumor-relate genes can be expressed by relieving the hypermethylation in promoter regions through inhibiting the expression of DNMT1 gene. It may provide a new target for gene therapy of human breast cancer.