Sperm penetration assay and in vitro fertilization experiment were performed to study the effects of the Afaf antibody on acrosome reaction and fertilization. Acrosome exocytosis (AE) with streptolysin O (SLO) permeabilization was conducted to test the Afaf's action in calcium events. Colocalization and coimmunoprecipitation was done to determine the interaction between Afaf and SNAP25 (synaptosome-associated protein of 25,000 daltons). Transferrin (Tf) uptake assay was performed to demonstrate the impact of Afaf on endosomal pathway. RNAi was used to rescue the inhibition of Afaf on Tf uptake.