We aimed to evaluate the effect of prednisolone (PDS) on natural killer cell (NK cell) cytolytic activity in vitro. Methods Blood samples from 74 patients with history of unexplained recurrent spontaneous abortion who elevated peripheral blood NK cell fraction were collected prospectively. Peripheral blood monocytes which containing NK cells were isolated and separated to three different tubes which containing target (K562) cells by the 50:1 effector to target (E:T) ratio. PDS or intravenous immunoglobulin (IVIG) was additionally added to 2 tubes for evaluate their suppressive effect. The percentage killing of target cells was recorded numerically by using flow cytometer and the values between groups were statistically analyzed. Results The mean target cell killing percentage was 40.5% in co-culture tube which was not added PDS or IVIG. In culture experiments which was added IVIG, the killing percentage is reduced to 37.7% which showed no significant differences compared to that of co-cultured tube which was not added PDS or IVIG. But, in experiments with added PDS, the killing percent was reduced to 19.5% and the difference was statistically significant ( P<0.001) compared to that of co-cultured tube which was not added PDS or IVIG. On comparing the reduction in killing percentage of target cells by PDS and IVIG, statistically significant reduction in the PDS coculture was noted ( P<0.005). Conclusion NK cell cytolytic activity is effectively down-regulated by using PDS in vitro. Moreover, the effect of PDS in down-regulation of NK cell cytolytic activity is seems to superior than that of IVIG. But, large scaled in vivo study is needed.