Rnd3, a Rho GTPase, is involved in the inhibition of actin cytoskeleton dynamics through the Rho kinase-dependent signaling pathway. We previously demonstrated that mice with genetic deletion of developed a markedly larger brain compared with wild-type mice. Here, we demonstrate that knockout mice developed an enlarged subventricular zone, and we identify a novel role for Rnd3 as an inhibitor of Notch signaling in neural stem cells. deficiency, both and , resulted in increased levels of Notch intracellular domain protein. This led to enhanced Notch signaling and promotion of aberrant neural stem cell growth, thereby resulting in a larger subventricular zone and a markedly larger brain. Inhibition of Notch activity abrogated this aberrant neural stem cell growth.