Objective: The relationship between peripheral blood Natural Killer (NK) Cells and pregnancy outcome after ICSI treatment has been examined in several studies, and their effects on reproductive failure is still controversial. The aim of this study was to evaluate the effect of the peripheral NK Cells, including total CD16+ CD56+ and activation marker CD69+ on pregnancy outcome after ICSI treatment. Design: This prospective observational study of 312 randomly selected women who underwent ICSI treatment from Jan. 2008 to Jan. 2010. Materials and Methods: Blood samples were obtained on the day of oocytes retrieval prior to the procedure. The CD16+ CD56+ NK Cells, CD69+ Activated NK Cells and CD19+ B"Cells were identified by flow cytometry. The results were presented as a percentage of total Lymphocytes, and their relationship to ICSI outcome were analyzed. Results: Receiver operating characteristic (ROC) curve and area under curve (AUC) analysis were performed to select CD16+ CD56+ and CD69+ thresholds for further statistical analysis. The clinical pregnancy rate (CPR) was significantly lower (P <0.0001) for women with CD16+ CD56+ NK Cells percentage more than 18% (CPR was 25%), compared with percentage below this value (CPR was 52.38%), and the serum CD16+ CD56+ NK cells demonstated a sensitivity of 68.3 (95% confidence interval [CI]:60.5–75.4) and specificity of 91.67 (CI: 85.9 – 95.62) for the prediction of implantation failure in ICSI patients. Moreover, CPR was also significantly lower (P <0.0001) when the percentage of activated CD69+ NK Cells was more than 1% (CPR was 27.54), compared with the percentage below this value (CPR was 54.11), and the sensitivity was 72.3 (CI: 59.5–77.4) and specificity of 86.81 (CI: 80.16–91.87) for the prediction of implantation failure in ICSI patients. Conclusions: The present study indicates that increase in the percentage of CD16+ CD56+ NK Cells or activated CD 69+ NK Cells in the peripheral blood is associated with a reduced implantation and clinical pregnancy rate in ICSI treatment. Keywords : NK Cells, ICSI, Activated CD69+ and Implantation failure.