Nesprins, also known as Nuance and Enaptin, are extremely large actin-binding proteins associated with the inner and outer nuclear membranes. Nesprins contain N-terminal actin binding domain (ABD) followed by coiled coil rod domain and the C-terminal transmembrane domain. Previous studies showed that the His-tagged ABD of Nesprin-1 binds and cross-links actin filaments into tight bundles. We have reexamined its actin-interaction properties using tag-free ABD and found that it binds to actin filaments without affecting their polymerization rate, but does not promote filament bundling. ABD also quenched the fluorescence of pyrene-labelled actin upon binding in a dose-dependent manner. This effect was used to determine the binding kinetics by stopped flow, and the Kd of the ABD.F-actin complex was estimated as 3.1 µM. Next, we assayed the ABD/actin interaction in the presence of myosin and found that the ABD disrupts the actomyosin complex in a dose dependent manner. Competitive binding of the ABD and myosin to actin filaments was also measured by light scattering during actomyosin dissociation after flash photolysis of caged ATP. ABD efficiently displaced myosin from actin filaments without affecting kinetics of myosin dissociation. Furthermore, ABD efficiently blocked motility of actin filaments in the myosin-based assay, partially dislodging them from the myosin-coated surface.