After completion of the acrosomal exocytosis, most of the inner acrosomal membrane becomes exposed, and these interactions end in fertilization (32). Mammalian spermatozoa must undergo AE before penetration of oocyte at fertilization (33). As Afaf is present on the inner and outer acrosomal membrane, we hypothesized that Afaf may be important in AE. Therefore, the anti-Afaf IgG was introduced for examining this possibility. We demonstrated that anti-Afaf IgG could influence AE and affect sperm-ZP penetration. It is suggested that Afaf may be involved in the fusion of the outer acrosomal membrane and plasma membrane.