Establishment of neural circuits in the central nerve system requires generation and development of multiple dendrites and single axon. Cultured neurons that showed sequence of morphological changes are well studied for neuronal morphogenesis. Ezrin/Radixin/Moesin (ERM) proteins link between membrane proteins and actin cytoskeleton, contributes to maintenance of cellular function and morphology. In cultured hippocampal neurons, suppression of both radixin and moesin showed deficits in growth cone morphology and neurite extensions. On the other hand, down-regulation of ezrin using siRNA caused impairment of netrin-1-induced axon outgrowth in cultured cortical neurons. However, roles of ezrin in the neuronal morphogenesis of the cultured neurons have been poorly understood. In this report, we performed detailed studies on the roles of ezrin in the cultured cortical neurons prepared from the ezrin knockdown (Vil2^kd/kd) mice embryo that showed a very small amount of ezrin expression compared with the wild-type (Vil2^+/+) neurons. Ezrin was mainly expressed in cell body in the cultured cortical neurons. We demonstrated that the cultured cortical neurons prepared from the Vil2^kd/kd mice embryo exhibited impairment of neuritogenesis, not neurite and axon outgrowth. Moreover, we observed increased RhoA activity and phosphorylation of myosin light chain 2 (MLC2), as a downstream effector of RhoA in the Vil2^kd/kd neurons. In addition, treatment of Y-27632, an inhibitor specific for Rho kinase, rescued the abnormalities in neuritogenesis in the Vil2^kd/kd neurons. These data altogether suggest a novel role of ezrin in the neuritogenesis of the cultured cortical neurons via down-regulation of RhoA/Rho kinase/MLC2 pathway.