Background: Chemical activation is the most frequently used method for artificial oocyte activation (AOA), results in high fertilization rate. Objective: To evaluate the efficiency of oocyte activation with calcium ionophore on fertilization and pregnancy outcomes after intracytoplasmic sperm injection (ICSI) in patients with previous fertilization failure. Design: prospective controlled study Materials and methods: One hundred and eight patients with history of previous fertilization failure undergoing IVF/ICSI treatment with long agonist protocol were randomly divided into two groups: group A (n=54) and group B (n=54) ; a total of 756 metaphase II (MII) oocytes were retrieved . In the oocytes of group A(n=350 oocyte), routine ICSI was applied; while in oocytes in group B (n=406 oocyte) immediately after ICSI, were entered in culture medium supplemented with 5 ?Μ calcium ionophore (A23187) for 10 minutes and then washed at least five times with MOPS solution. Main outcome measures: In both groups, the fertilization was evaluated after 16-18 hours. Results: The number of fertilized oocytes and embryos obtained were significantly different between two groups (p=<0.001*). Fertilization rate was significantly higher in group B ?where calcium ionophore was applied-compared to group A(control group) (32.2% vs. 9.1%, respectively ,p=0.01*); and Cleavage rate also was significantly higher in group B compared to group A (27.4% vs 12.5% respectively, p=0.028*). Implantation rate was significantly higher in group B than in group A (18.32% vs. 3.12 % respectively, p=0.035*) Pregnancy rate also was significantly higher in group B than in group A (22.2% vs. 1.85% respectively, p=0.042*). Conclusion: Chemical oocyte activation with calcium ionophore resulted in a significant improvement in fertilization, cleavage, implantation and pregnancy rates after ICSI in infertile patients with previous fertilization failure.