NK cell cytotoxicity requires the formation of an actin-rich immunological synapse (IS) with a target cell and the polarization of perforin-containing lytic granules toward the IS. Following the polarization of lytic granules, they traverse through the actin-rich IS to join the NK cell membrane in order for directed secretion of their contents to occur. We examined the role of myosin IIA as a candidate for facilitating this prefinal step in lytic NK cell IS function. Lytic granules in and derived from a human NK cell line, or ex vivo human NK cells, were constitutively associated with myosin IIA. When isolated using density gradients, myosin IIA-associated NK cell lytic granules directly bound to F-actin and the interaction was sensitive to the presence of ATP under conditions of flow. In NK cells from patients with a truncation mutation in myosin IIA, NK cell cytotoxicity, lytic granule penetration into F-actin at the IS, and interaction of isolated granules with F-actin were all decreased. Similarly, inhibition of myosin function also diminished the penetration of lytic granules into F-actin at the IS, as well as the final approach of lytic granules to and their dynamics at the IS. Thus, NK cell lytic granule-associated myosin IIA enables their interaction with actin and final transit through the actin-rich IS to the synaptic membrane, and can be defective in the context of naturally occurring human myosin IIA mutation.