The NK immunological synapse (NKIS) is a dynamic structure dependent on the assembly of membrane, cytoskeletal and signaling components. These serve to focus and generate stimuli for adhesion and orientation of the cytoskeleton for targeted cytolytic granule release. Previous studies have demonstrated the importance of the cytoskeleton in these processes. We previously identified PPP1R9B (neurabin 2, spinophilin) as a cytoskeletal component of the NK-like cell line YTS. We demonstrate that (i) PPP1R9B gradually accumulates at the NKIS in a maturation stage-dependent manner; (ii) it mimics the early kinetics of actin recruitment to the NKIS but it precedes actin departure from the site; (iii) it is recruited by CD18 stimulation but not by CD28 ligation; (iv) it is required for the maintenance of the cortical F-actin organization in the YTS cells and knocking down PPP1R9B reduces the frequency of YTS-target cell conjugation, possibly due to the collapsed F-actin cytoskeleton in these cells. These results indicate that PPP1R9B is required for synapse formation in the NK cells and suggest that it may be involved in the maintenance of cellular architecture by regulation of actin assembly, possibly acting to stabilize the NKIS until granule release is eminent.