Natural killer (NK) cells play important roles in host immunity by killing virus-infected and tumor cells. Killing of the target cell is achieved by formation of an immune synapse and localized secretion of lytic granules containing perforin and granzymes. Here, we demonstrate that Wiskott-Aldrich syndrome protein (WASp)-interacting protein (WIP), important in generation of a large complex of proteins involved in actin cytoskeleton rearrangements, is indispensable for NK cell cytotoxicity. WIP knockdown completely inhibited cytotoxicity, whereas overexpression of WIP enhanced NK cell cytolytic ability. WIP was found to colocalize with lytic granules. WIP segregated to the lysosomal fraction, where granzyme B activity was also found, and the interaction between WIP and granules was independent of WASp. Importantly, WIP knockdown inhibited polarization of lytic granules to the immune synapse, but not conjugate formation. These results indicate that WIP is involved in lytic granule transport and is essential for regulation of NK cell cytotoxic function.