Objective: To construct the STAT3 shRNA (short hairpin RNA) lentivirus and investigate the effects of STAT3 SiRNA(small interference RNA)on the biological property of human chronic myelogenous leukemia (CML) K562 cells. Methods:The STAT3 shRNA lentiviral vector was constructed according to the effective target sequences for RNAi of STAT3 gene. The STAT3 SiRNA lentivirus were produced by transfecting into packaging cell line 293T. After transfected into K562 cells, the knockdown of STAT3 gene were determined by RT-PCR and Western-blot assay. The survival and proliferation of K562 cells were detected by both cell growth curve and MTT assay. The FCM was used to detected cell cycles alternation and the early apoptosis by Annexin V-PI assay. Results:PCR and sequencing analysis verified that the construction of the lentiviral vector of STAT3 shRNA was successful and the expression of STAT3 gene was knockdowned around 70 percent by two target sequences for STAT3 RNAi in K562 cells. Proliferation of K562 cells became slower and the proliferation activity of K562 cell was decreased to 40% post-transfection 5 days by lentiviral-mediated STAT3 SiRNA(P < 0.05). The proportion of cells in G0/G1 phase were increased while the cells in S phase decreased, which indicated a delay at G1 to S checkpoint in cell cycles. More than half the K562 cells underwent apoptosis after transfected with STAT3 SiRNA lentivirus compared to the control cells[(51.80±5.60)% vs (1.13±0.12)%],respectively(P < 0.01). Conclusion:Lentivirus-based STAT3 SiRNA can effectively mediate STAT3 gene silencing in K562 cells. SiRNA targeting STAT3 gene can inhibit K562 cell growth and proliferation, block cell cycle at G1 to S phase and induce cell apoptosis.