Class-18 myosins challenge our established view about myosins acting as molecular motors. No member of this class appears to have a significant ATPase activity, which is a prerequisite for motor activity. Humans express two myosin-18 isoforms, myosin-18A and myosin-18B. Whereas recent studies on myosin-18A shed some light on its cellular and biochemical mode of action, the molecular function of myosin-18B remains poorly understood. Class-18 myosins contain protein interaction domains outside their generic motor domain. In the case of myosin-18B this includes a large, N-terminal extension that shows no similarity to any known protein domain. Here, we show that the human myosin-18B motor domain binds to F-actin with an affinity of 4 µM. The isolated motor domain binds ATP but has no intrinsic ATPase activity. The large N-terminal extension is shown to directly bind to F-actin with an affinity of 7 µM. This interaction is nucleotide-independent but shows strong ionic strength dependence, which is indicative for a charge-mediated actin binding mechanism. We further analyzed the molecular function of the N-terminal extension by means of actin polymerization assays and found that the myosin-18B N-terminus inhibits F-actin assembly in vitro. Myosin-18B has previously been shown to be located in the cytoplasm of undifferentiated myoblasts. At later stages of differentiation it accumulates in myonuclei. Furthermore, it has been shown that cardiomyocytes display a partial sarcomeric pattern of myosin-18B alternating that of α-actinin-2. Based on our data, we propose a role for myosin-18B in the regulation of muscle sarcomere architecture during differentiation and the regulation of the nuclear actin pool.