Abstract: Objective: To investigate the cytotoxicity of cytokine-induced killer (CIK) cells against human Esophageal Carcinoma cell line EC9706, in vitro. Methods:The expression of NKG2D ligands of EC 9706cells were analyzed by flow cy-tometry. CIK cells were generated from peripheral blood mononuclear cells of healthy donors by culturing cells in the pres -ence of IFN-γ, anti -CD3 antibody and IL-2, in vitro. The percentage of CD 3+ CD56+ and the expression of NKG2D in the bulk CIK cells at days 0 and 14were analyzed by flow cytometry. Cytotoxicity of CIK cells against EC9706 cells was mea-sured using a standard LDH releasing assay. The effector cells were added to the target cells at E:T ratios of 10:1, 20: 1, 30:1, 40:1 and 50:1. In blocking experiments, NKG2D monoclonal antibody was added to CIK cells for 15min before plat-ing at an E:T ratio of 30:1. Results: It was found that MIC and ULBP 2 were expressed; however MICB, ULBP1 and ULBP 3 were not detectable by EC 9706cells. At days 0 and 14, the percentage of CD3+ CD56+ cells were (0.68± 0.07)% and ( 56.55±2.01)%, respectively ( P<0.05), while the expression rates of NKG2D were ( 26.30± 1.12)% and ( 67.13± 1.34)%, respective -ly ( P<0.05). The cytotoxicity of CIK cells against EC9706 cells were (28.81± 0.47)% , ( 37.78± 0.22)% , ( 44.31± 1.06)% , (47.25± 0.47)% and (57.62± 0.94)% , respectively, at E:T ratios of 10:1, 20:1, 30:1, 40:1 and 50:1 (P<0.05). When the NKG2D molecules on bulk CIK cells were blocked by the NKG2D monoclonal antibody, the cytolytic activity of the CIK cell was significantly inhibited. Conclusion:During CIK cell expansion, NKG2D and CD3+ CD56+ are upregulated. NKG 2D mole-cules play a role for cytotoxicity against EC9706cells.