The interaction of ultra-nanocrystalline diamond (UNCD) with neural stem cells (NSCs) has been studied in order to evaluate its potential as a biomaterial. Hydrogen-terminated UNCD (H-UNCD) films were compared with standard grade polystyrene in terms of their impact on the differentiation of NSCs. When NSCs were cultured on these substrates in medium supplemented with low concentration of serum and without any differentiating factors, H-UNCD films spontaneously induced neuronal differentiation on NSCs. By direct suppression of mitogen-activated protein kinase/extracellular signaling-regulated kinase1/2 (MAPK/Erk1/2) signaling pathway in NSCs using U0126, known to inhibit the activation of Erk1/2, we demonstrated that the enhancement of Erk1/2 pathway is one of the effects of H-UNCD-induced NSCs differentiation. Moreover, functional-blocking antibody directed against integrin β1 subunit inhibited neuronal differentiation on H-UNCD films. This result demonstrated the involvement of integrin β1 in H-UNCD-mediated neuronal differentiation. Mechanistic studies revealed the cell adhesion to H-UNCD films associated with focal adhesion kinase (Fak) and initiated MAPK/Erk1/2 signaling. Our study demonstrated that H-UNCD films-mediated NSCs differentiation involves fibronectin-integrin β1 and Fak–MAPK/Erk signaling pathways in the absence of differentiation factors. These observations raise the potential for the use of UNCD as a biomaterial for central nervous system transplantation and tissue engineering.