The development of an efficient platform for the growth and neuronal differentiation of stem cells is crucial for autologous cell therapy and tissue engineering to treat various neuronal disorders and neurodegenerative diseases. In this study, we describe the use of highly uniform graphene platforms that provide unique environments where unusual three-dimensional spheroids of human mesenchymal stem cells (hMSCs) are formed, which is advantageous for the differentiation of hMSCs into neurons. We suppose that graphene regulates the interactions at cell-substrate or cell-cell interfaces, consequently promoting the neurogenesis of hMSCs as well as the outgrowth of neurites, which was evidenced by the graphene-induced upregulation of early neurogenesis-related genes. We also demonstrated that the differentiated neurons from hMSCs on graphene are notably sensitive to external ion stimulation, and their neuronal properties can be maintained even after detaching and re-seeding onto a normal cell culture substrate, suggesting the enhanced maturity of resulting neuronal cells. Thus, we conclude that monolayer graphene is capable of regulating the growth and neural differentiation of hMSCs, which would provide new insight and strategy not only for autologous stem cell therapy but for tissue engineering and regenerative medicine based on graphene scaffolds.