One of the most important processes in fertilization is the fusion of egg and sperm; however, the molecular mechanisms involved in this process are not well understood. So far, using genetic approaches, only two proteins have been demonstrated to be necessary for this process: Izumo in sperm and CD9 in the egg. Here we demonstrate that sperm produced by Tssk6 (Sstk)-null mice present defects that prevent the successful fertilization of eggs in vitro and the fusion to zona-pellucida-free eggs. Tssk6 is a member of the testis-specific serine kinase family of proteins and is expressed postmeiotically in male germ cells. In order for fusion to occur, during the process known as acrosome reaction Izumo needs to relocate from the anterior head to other regions, including the postacrosomal compartment. Tssk6-null sperm fails to relocate Izumo during the acrosome reaction. Agents that interfere with actin dynamics blocked the acrosome-reaction-associated translocation of Izumo that is required for fusion in wild-type sperm. Additionally, actin polymerization was compromised in Tssk6-null sperm. Taken together, our results indicate that Tssk6 is involved in sperm-egg fusion through the regulation of actin polymerization and changes in Izumo localization.