Objective To investigate sensitivity of colon cancer cells to 5-fluorouracil after downregulation of XIAP gene expression. Method Colon cancer cells HCT-8 and HCT116 were transfected with a short hairpin RNA targeted to XIAP by liposome, cells viability were examined.5-fluorouracil was applied into two kinds of colon cancer cells. Tumor cells sensitiviy to chemotherapeutic drug was evaluated. Caspase-3 activity in tumor cells was examined by Western blot. Result After downregulation of XIAP expression, cell growing viability of these two kinds of colon cancer cells was restricted, HCT-8 resistance to 5-fluorouracil was reversed ( P ＜ 0. 01 ), HCT116 sensitivity to 5-fluorouracil was enhanced (P ＜ 0.05), caspase-3 expression in colon cancer cells was highly activated, apoptosis inducing activity of 5-fluorouracil was increased significantly. Conclusions XIAP expression was a important mechanism in colon cancer cells HCT-8 and HCT 116 resistant to 5-fluorouracil, sensitivity to 5-fluorouracil of HCT-8 and HCT-116 was increased by downregulation of XIAP expression.