RAC1 encodes a Rho family small GTPase that regulates actin cytoskeletal reorganization and intracellular signaling pathways. Variants in RAC1 can cause a phenotypically heterogeneous neurodevelopmental disorder with brain size anomalies and facial dysmorphism. Pathophysiological mechanism of this genetic disorder remains to be clarified. We investigated an early adolescent male with global developmental delay, intellectual disability, and secondary microcephaly. The novel de novo variant (NM_006908.5): c.92A>G, p.(E31G) in RAC1 was identified by whole exome sequencing. We then examined the pathophysiological significance of this variant by focusing on brain development using in vitro and in vivo methods. The p.E31G variant had little and slight effects on the intrinsic GTP/GDP exchange and GTP-hydrolysis activity in vitro, respectively, while it was reactive normally to a guanine-nucleotide exchange factor and a GTPase-activating protein. Meanwhile, the variant did not bind to its downstream effector PAK1 even in the GTP-bound status, and suppressed the differentiation of primary cultured hippocampal neurons weakly when overexpressed. In vivo analyses using in utero electroporation displayed that acute expression of the p.E31G variant in the young adult stage caused defects in dendritic arbor development and axon extension of cortical neurons, whereas it had little effects on neuron migration during corticogenesis. Our results indicate that RAC1 is critical for brain development. The p.E31G variant is most likely to serve as a dominantly negative version to PAK1-mediated signaling pathway. This may be responsible for morphological and functional defects in cortical neurons, leading to the clinical features of the present patient.