To explore ZO-1 gene expression and methylation in leukemia cells and the involvement of ZO-1 gene in leukemogenesis.Restriction landmark genomic scanning (RLGS) was used to identify new leukemia related gene, and methylation specific PCR (MSP) for ZO-1 methylation status. ZO-1 specific siRNA was designed and prepared by in vitro transcription and transfected into K562 cells, the transfected cells were cultured for 48 hours before harvesting. The effect of ZO-1 siRNA was monitored by Northern blot. Cellular proliferation capacity was assayed by CCK-8, cell apoptosis by Annexin V-fluorescence in isothiocyanate (FITC) assay, and cell cycle by phosphatidylinositol (PI).The intensified spots in RLGS gel were subjected to bioinformatics analysis and one of the candidate spots was proved to be ZO-1 gene. In fresh leukemia cells, Molt4 cells and HL-60 cells, ZO-1 was hypermethylated, causing it reduced or silenced. ZO-1 gene was highly expressed with no methylation in normal peripheral blood MNC and K562 cells. There was a good correlation between promoter methylation and the gene silence. The silenced gene can be re-activated by demethylation treatment with 5-AZA-dC in most leukemia cell lines. RNA interference for ZO-1 gene in K562 cells did not interfere with cell proliferation, cell cycle and apoptosis.ZO-1 gene methylation might be involved in the tumorigenesis of acute leukemia.