Abstract: OBJECTIVE To construct eukaryotic expression vectors of short hairpin RNA(shRNA) of STAT3 gene and investigate the effect of shRNA STAT3 on chemotherapy sensitivity of HCT116 cells to cisplatin. METHODS Plasmids carrying shRNA targeting STAT3 were designed constructed and transfected into HCT116 cells by liposome transfection methods. The expressing levels of STAT3 protein were detected by Western blot. Cell survival rate was observed with MTT. The role of plasmids in combination with cisplatin on the HCT116 cells was observe. The cell growth was assessed by MTT assay. RESULTS DNA sequence analysis demonstrated that the eukaryotic expression vector of pGPU6/GFP/Neo-STAT3 were constructed successfully. The recombinant plasmid was transfected into HCT116 cells, cell proliferation was obviously inhibited, reducing the expression of STAT3 protein. After plasmid and cisplatin combination treatment, cell survival rate was obviously decreased. CONCLUSION STAT3 shRNA plasmid could significantly down-regulate the expression of STAT3 protein in HCT116 cells, inhibit cell proliferation and improve the sensitivity to cisplatin.