In summary, our results suggest that Afaf as an acorosome membrane protein, may participate in AE and endocytic membrane trafficking, and therefore plays an important role in fertilization. However, the molecular mechanism of how Afaf affects exocytosis and endocytic trafficking is still not clear, and remains for further investigation. Our observation may also provide an additional evidence for the concept that the complex associated with SNARE proteins, at least SNAP25, are multifunctional, and may mediate distinct membrane-specific fusion reactions.