The Notch signaling pathway has been shown to be involved in the progression of chronic myeloid leukemia (CML). The aim of this study was to investigate the effects of exogenous Notch2 overexpression on cell proliferation and possible mechanisms in the human CML cell line K562. When exogenous intracellular fragment of Notch2 (ICN2) was transfected into K562 cells with Lipofectamine™ 2000, the expression of Notch2 mRNA and protein were upregulated. Cell numbers decreased and the proliferation was inhibited significantly after transfection with ICN2. G1 phase cells increased and S phase cells decreased 48 h after transfection. Finally, the expression of Numb, Bcl-2, NF-κB and TGF-β1 was detected. It was found that the expression of NF-κB and TGF-β1 mRNA was increased, while Bcl-2 was downregulated, with Numb expression unchanged. Our study indicates that the Notch pathway is activated in K562 cells after ICN2 transfection. It inhibited the proliferation of K562 cells, likely by upregulating the expression of NF-κB and TGF-β1 mRNA and downregulating the expression of Bcl-2.