Objective: To study the effects of down-regulation of HDAC6 expression on proliferation, cell cycling and migration of esophageal squamous cell carcinoma (ESCC) cells and related molecular mechanisms. Methods: ESCC cell line EC9706 cells were randomly divided into untreated (with no transfection), control siRNA (transfected with control siRNA) and HDAC6 siRNA (transfected with HDAC6 small interfering RNA) groups. Effects of HDAC6 siRNA interference on expression of HDAC6 mRNA and protein in EC9706 cells were investigated by semi-quantitative RT-PCR, Western blotting and immunocytochemistry methods. Effects of down-regulation of HDAC6 expression on cell proliferation, cell cycle, and cell migration were studied using a CCK-8 kit, flow cytometry and Boyden chambers, respectively. Changes of mRNA and protein expression levels of cell cycle related factor (p21) and cell migration related factor (E-cadherin) were investigated by semi-quantitative RT-PCR and Western blotting methods. Results: After transfection of HDAC6 siRNA, the expression of HDAC6 mRNA and protein in EC9706 cells was significantly downregulated. In the HDAC6 siRNA group, cell proliferation was markedly inhibited, the percentage of cells in G0/G1 phase evidently increased and the percentage of cells in S phase decreased, and the number of migrating cells significantly and obviously decreased. The mRNA and protein expression levels of p21 and E-cadherin in the HDAC6 siRNA group were significantly higher than those in the untreated group and the control siRNA group, respectively. Conclusions: HDAC6 siRNA can effectively downregulate the expression of HDAC6 mRNA and protein in EC9706 cells. Down-regulation of HDAC6 expression can obviously inhibit cell proliferation, arrest cell cycling in the G0/G1 phase and reduce cell migration. The latter two functions may be closely related with the elevation of mRNA and protein expression of p21 and E-cadherin.