The sperm protein IZUMO1 plays a central role in gamete fusion. In mouse sperm, IZUMO1 is enriched at the acrosomal cap before the acrosome reaction, and at the equatorial segment following this reaction; its relocation is dependent on filamentous actin. How actin polymerization affects IZUMO1 relocation during gamete interaction remains unknown. The present study addressed these processes using latrunculin A (LatA), an inhibitor of actin polymerization. We report that 25 µM LatA blocked actin polymerization in the capacitated sperm head, resulting in a marked decrease in sperm with relocated IZUMO1 during the A23187-induced acrosome reaction and cumulus layer penetration. Treated sperm also exhibited reduced zona pellucida penetration and fertilizing capacity. Interestingly, LatA-treated sperm present in the perivitelline space of eggs did not show impaired IZUMO1 relocation. Thus, IZUMO1 relocation represents one method by which eggs may select for or rescue sperm that are competent to undergo gamete adhesion/fusion. These data support the hypothesis that dynamic movement of IZUMO1 is essential for gamete fusion during mouse fertilization.