Neurons are highly polarized cells, with long axons and branched dendrites that define their architecture and function. Achieving this polarized state requires dynamic cytoskeleton remodeling, targeted protein trafficking, and membrane delivery to specific growth sites, processes largely coordinated by the Rho family of small GTPases. Unlike the well-characterized RhoA, Rac1, and Cdc42, RhoD exhibits unusually high intrinsic GDP/GTP exchange activity, classifying it as a fast-cycling GTPase. Expressed exclusively in mammals, RhoD has been implicated in regulating actin cytoskeleton dynamics, Golgi organization, endosome motility, cell migration, and axon guidance. The aim of this study is to unveil the role of RhoD in neuronal polarity and development. We found that silencing RhoD via shRNA in hippocampal neuron cultures altered stage transitions and neurite outgrowth, resulting in longer axons in early cultures and reduced dendritic complexity in more mature neurons. Expression of RhoD activity mutants impaired neurite extension and disrupted neuronal migration during cortical development in situ. These observations indicate that RhoD is a critical regulator of cytoskeletal dynamics and neuronal differentiation. Additionally, we developed and characterized a FRET-biosensor to analyze the spatiotemporal dynamics of RhoD activation in living neurons.