Natural killer (NK) cells are lymphocytes of the innate immune system that provide defense by directing their cytotoxic activity against virally infected and tumorigenic cells. Target cell lysis requires directed secretion of lytic granule contents, including pore-forming perforin and apoptosis-inducing granzymes, at the contact site. During the formation of a mature immunological synapse (IS) with a target cell, an NK cell polymerizes filamentous actin (F-actin) at the contact site, which may serve as a barrier to secretion of lytic granules for cytotoxicity. An actin-based motor protein, nonmuscle myosin IIA, has been demonstrated to be required for NK cell cytotoxic activity. In this work, we examine the role of myosin IIA in NK cell cytotoxicity. Through studies of primary human NK cells and NK cell lines, we find that myosin IIA associates with NK cell lytic granules. This functional association is required for NK cell granule exocytosis because myosin IIA mediates an interaction of lytic granules with F-actin at the IS. We find that NK cell cytotoxicity is impaired in patients with mutations in the myosin IIA heavy chain, and that mutations in the motor domain and nonhelical tailpiece specifically affect NK cell function. Finally, we demonstrate that single molecules of myosin IIA associate with NK cell lytic granules via the nonhelical tailpiece of the myosin IIA heavy chain, and that this interaction requires phosphorylation of the tailpiece at Ser1943. Thus, we present a novel role for nonmuscle myosin IIA in NK cells, where myosin IIA directly associates with lytic granules and guides their transport across actin filaments to facilitate secretion of their contents.