This study was aimed to explore the effect of DLL4/Notch1 ligand on cell growth in leukemia cell line K562 and its relevant mechanism. The pBudCE4.1-DLL4 plasmid was transfected into K562 cells by lipofectamine 2 000, RT-PCR and Western blot were applied to monitor the mRNA and the protein expression of exogenous DLL4 gene, as well as the expression of Notch1-ICD and target gene Hes1. Expression levels of Rb, YY1 and C-MYC protein in K562 cells were also detected by Western blot. Cell counting Kit-8 was used to detect the proliferation of K562 cells, and flow cytometry with Annexin V staining was used to detect the cell apoptosis. The results showed that the mRNA and protein expression levels of DLL4, Notch1-ICD and Hes1 in cells of experimental group were significantly higher than those of control groups (P < 0.05), indicating the successful activation of the Notch1 signaling pathway. The protein expression levels of Rb, YY1 and C-MYC in cells of experimental group significantly increased when compared with that of control group cells (P < 0.05). After transfection, the proliferation of K562 cells was obviously inhibited, and apoptosis rate in DLL4-transfected cells was significantly enhanced. DLL4 transfection significantly increased the number of cells in G1 phase and decreased that in S phase. It is concluded that the over-expression of DLL4 ligand gene in K562 cells results in successful activation of the Notch1 signaling pathway, increases expression of Rb, YY1 and C-MYC genes, which induces apoptosis and reduces proliferation.