Fertilization failure after ICSI may be due to the inability of the spermatozoa to trigger oocyte activation which, is characterized by a rise in intracellular calcium concentration. Treatment with calcium ionophore may increase the free intracellular calcium, thereby mimicking physiological cell-signaling mechanisms resulting in oocyte activation.The aim of this study was to evaluate the impact on reproductive outcome of oocyte activation (OA) by calcium ionophore (A23187) in patients with complete fertilization failure following ICSI during previous In Vitro Fertilization (IVF) cycles. Prospective cohort study. Between January 2011 and December 2013 30 couples who had prior insuccessful ICSI treatments due to complete fertilization failure accepted to enter this study. Following ICSI 217 injected oocytes were activated with calcium ionophore A23187 for twenty minutes. Embryo transfer was performed on day 3. Fertilization and top quality embryos rate, implantation, clinical pregnancy and birth rates were analyzed. Oocyte retrieval in 30 patients resulted in 288 cumulus-oocyte complexes. After denudation, 217 (84.3%) were mature and, consequently, available for ICSI. Fertilization rate was 63.1% (137/217), cleavage rate was 92% (126/137). A mean of 2.9 Day 3 embryos were transferred to 26 patients. Four couples had no embryos to transfer. Clinical pregnancy rate was 38.5% (10/26), miscarriage rate was 20% (2/10). Nine healthy babies were born (one twin pregnancy). The clinical use of ionophores in assisted reproduction is still limited by insufficient knowledge about their potential toxic effect on oocytes and embryos. However, healthy children already born seem not to be affected by ionophore treatment in our study. Assisted oocyte activation with calcium ionophore may become a reasonable and efficient treatment in cases of very low or complete fertilization failure. More prospective controlled studies should be performed to confirm these results.