This study was purposed to investigate the expression and role of eukaryotic expression vector containing p16, dll4 genes in leukemia K562 cells. A vector pBudCE4.1-16-dll4 containing wild type p16cDNA and dll4cDNA was designed and constructed, then this vector was transfected into leukemia K562 cells by using lipofectamine 2000. The expression of p16 and dll4 genes was detected by Western blot, the cell growth curve and cell cycle were determined by CCK-8 kit and flow cytometry respectively. The results showed that the recombinant plasmid pBudCE4.1-16-dll4 was constructed and transfected into K562 cells in vitro successfully. The expression of exogenous P16 and Dll4 proteins could be detected in K562 cells. After transfection for 48 hours, the K562 cells were arrested in G(1) phase, the cell count increased in G(0)/G(1) phase and reduced in S phase, the cell proliferation decreased as compared with control. It is concluded that the p16 and dll4 genes can simultaneously express in K562 cells transfected with recombinant plasmid pBudCE4.1-16-dll4 in vitro which results in G(0)/G(1) arrest and reduces cell proliferation.