Background: Extensive trials have indicated that cancer cells with high glycolytic activity exhibit decreased sensitivity to anticancer agents. Moreover, recent research has proven that specific inhibitors of hexokinase (HK) II, a key glycolytic enzyme, may enhance the activity of anticancer drugs. The aim of this study was to investigate the effect and mechanisms of HK II on chemosensitivity of a colon cancer cell line (LoVo) to 5-fluorouracil (5-FU). Methods: HK II gene expression was downregulated by RNA interference in the colon cancer cell line LoVo, which was detected by Western blot analysis. Then the IC<sub>50</sub> value of 5-FU was determined in LoVo cells via MTT assay. In addition, cell apoptosis and mitochondrial membrane potential (MMP) were assessed by flow cytometry and caspase-3 activity by its substrate color reaction. Results: In LoVo cells, HK II downregulation resulted in a decreased IC<sub>50</sub> value of 5-FU and increased apoptosis. Furthermore, HK II downregulation resulted in a decreased MPP and activation of caspase-3. Conclusion: Our findings suggest that targeting HK II may be beneficial for patients with colon cancer treated with 5-FU.