Objective To investigate the effect of different numbers of target cells on the cytotoxicity of NK-92MI against target cells at the same effector-to-target（ E∶T） ratio and its potential underlying mechanisms. Methods Under standard 4-hour cytotoxicity conditions， NK-92MI cells were co-cultured with K562， BNI 2-4-S， BNI 1-3-S， and BNI 19-1-S cells in quantities of 20 000， 10 000， 5 000， and 2 500 cells， respectively， and the lysis ratio of target cells was detected to determine the cytotoxic efficacy of NK-92MI. NK-92MI was coupled with K562 and BNI 19-1-S cells in quantities of 2 500， 5 000， 10 000， and 20 000 to investigate the binding of NK-92MI to target cells. Results In the cytotoxicity assay， when the number of target cells was 2 500， 5 000， 10 000， and 20 000， the lysis ratios of NK-92MI co-cultured with K562 cells were（ 16.62±2.09）%，（ 20.84±1.36）%，（ 31.00±2.01）%， and（ 47.80±1.30）%， respectively； the lysis ratios of NK-92MI co-cultured with BNI 2-4-S cells were（ 29.94±2.27）%，（ 39.64±2.47）%，（ 49.29±1.27）%， and（ 64.25±1.92）%， respectively； the lysis ratios of NK-92MI co-cultured with BNI 1-3-S cells were （41.81±3.59）%，（ 48.59±3.41）%，（ 57.10±3.25）%， and（ 64.55±1.90）%， respectively； the lysis ratios of NK-92MI co-cultured with BNI 19-1-S cells were（ 41.39±2.39）%，（ 47.85±3.15）%，（ 54.09±0.67）%， and （57.82±2.48）%， respectively. The cytotoxic efficacy of NK-92MI increased with the number of target cells， with statistically significant differences（ all P＜0.01）. In the cell-coupling assay， when the target cell numbers were 2，500， 5，000， 10，000， and 20 000， the binding ratios of NK-92MI to K562 cells were（ 1.85±0.11）%， （5.30±0.37）%，（ 7.79±0.08）%， and（ 13.10±0.30）%， respectively. In the cell coupling assay， when the target cell numbers were 2 500， 5 000， 10 000， and 20 000， the binding ratios of NK-92MI to K562 cells were （1.85±0.11）%，（ 5.30±0.37）%，（ 7.79±0.08）%， and（ 13.10±0.30）%， respectively； and the binding ratios of NK-92MI to BNI 19-1-S cells were（ 2.58±0.21）%，（ 6.69±0.31）%，（ 8.46±0.20）%， and（ 13.37±0.78）%， respectively. As the number of target cells increased， the binding of NK-92MI to target cells significantly increased， with statistically significant differences（ all P ＜ 0.01）. Conclusions At the same E ∶ T ratio， varying numbers of target cells influence the degree of contact between NK-92MI and target cells， thereby affecting the cytotoxic efficacy of NK-92MI against them. At the same E：T ratio， the greater the number of target cells， the more extensive the contact between NK-92MI and target cells， and the higher the cytotoxic efficacy of NK-92MI against them.