Introduction: The aim of this study was to evaluate the effect of sperm chromatin anomalies on fertilization outcome post ICSI. Material and Methods: Therefore along with semen parameters, CMA3 staining for protamine deficiency, aniline blue staining for excessive histones, SDS for sperm chromatin stability and SDS+EDTA for ability of sperm to undergo decondensation were carried out on 55 semen samples from patients referring for ICSI to Isfahan fertility and infertility cente. Result: The results showed that among the aforementioned tests and semen parameters, only CMA3 showed a significant correlation with fertilization outcome post ICSI. Patients were also grouped according to CMA3 level of <30% or >30 % or fertilization rate of <50%, or >50%. The results showed that the mean percentage fertilization and mean percentage of CMA3 positivity is different inboth groups respectively. The area under ROC curve showed that CMA3 is a highly sensitive and specific test for prediction of fertilization outcome post ICSI. Conclusion: Therefore, it can be concluded that sperm protamine deficiency has profound effect on fertilization failure in ICSI.