We report the cytocompatibility and regulating effects of the nanostructured reduced graphene oxide (rGO) microfibers, which are synthesized through a capillary hydrothermal method, on neural differentiation of neural stem cells (NSCs). Our findings indicate that the flexible, mechanically strong, surface nanoporous, biodegradable, and cytocompatible nanostructured rGO microfibers not only offer a more powerful substrate for NSCs adhesion and proliferation compared with 2D graphene film and tissue cluture plate but also regulate the NSCs differentiation into neurons and form a dense neural network surrounding the microfiber. These results illustrate the great potential of nanostructured rGO microfibers as an artificial neural tissue engineering (NTE) scaffold for nerve regeneration.