Abstract: Objective The aim of this study was to construct eukaryotic expression plasmid for human pCMV-DNA methyltransferase 3B7 (DNMT3B7) and investigate the effects of over-expression of DNMT 3B7 gene on proliferation of human embryo kidney 293A cells. Methods DNMT3B7 was amplifi ed by PCR using highfi delity Taq and cloned into pCMV-2B. 293A cells were transfected with plasmid pCMV-DNMT 3B7 or pCMV-2B) and then treated with G418 to get the stable cell lines. The proliferation level of stable cell lines was determined by MTT, and cell cycle distribution by fl ow cytometry. Results DNMT3B7 eukaryotic expression plasmid was successfully constructed. Compared with those 293A cells transfected with pCMV-2B, the number of those cells with over-expression DNMT 3B7 was decreased (P＜0.01). The percentage of 293A cells with overexpression DNMT 3B7 at S stage was decreased (P＜0.05). The mRNA level and protein level of p21 were increased (P＜0.05). Conclusion The over-expression of DNMT 3B7 gene inhibited the proliferation of 293A, maybe through p21.