This study was aimed to explore the effects of Notch ligand DLL4 on the protein expression of the transcription factor YY1 and proto-oncogene c-Myc, as well as K562 cell proliferation. The experiment was divided into 3 groups: normal control, negative control (pBudCE4.1-transfected) and experimental (pBudCE4.1-DLL4-transfected) groups. At 48 hours after transfection, the expression level of DLL4, YY1 and c-Myc proteins in K562 cells of each group were detected by Western blot and indirect immunocytochemical method; the CCK-8 method was used to detect proliferation of K562 cells; at 48 hours after transfection, cell cycle distribution and apoptosis of K562 cells were detected by flow cytometry. The results showed that the protein expression of DLL4, YY1 and c-Myc in K562 cells of every group were found. The protein expression levels of DLL4, YY1 and c-Myc in the experimental group cells were significantly higher than that in control groups (p < 0.05). The cell number in G(0)/G(1) phase increased in the experimental group and was higher than that in the control groups (p < 0.001), and the number of apoptotic cells were also increased (p < 0.001). It is concluded that DLL4 gene was successfully transfected into K562 cells, which increased the protein expression levels of transcription factor YY1 and proto-oncogene c-Myc, leading to the cell proliferation slower in experiment group, inducing the cell cycle arrested in G(0)/G(1) phase and increasing apoptosis.