Neural stem cells (NSCs) are deemed to be a potential cell therapy for brain and spinal cord reconstruction and regeneration following injury. In this study, we investigated the role of nanofibrous scaffolds on NSCs-derived neurons in the formation of neural networks. Miniature excitatory postsynaptic currents (mEPSCs) were recorded using the whole-cell patch clamp recording method after the spinal cord-derived NSCs were differentiated into neurons and cultured in vitro for 10-14 days. It was observed that the frequency of mEPSCs in the differentiated neurons cultured on both randomly oriented and aligned collagen nanofibrous scaffolds was higher than that on the collagen-coated control and can be inhibited by an ERK inhibitor (PD98059), indicating that the collagen nanofibers affected the maturation of the synapses from presynaptic sites via the MAPK/ERK1/2 pathway. In addition, both of the collagen nanofibers increased the phosphorylation of Synapsin I and facilitated the interaction of p-ERK1/2 and p-Synapsin I. All these results suggested that the collagen nanofibrous scaffolds contributed to the presynaptic maturation via the ERK1/2-Synapsin I signaling pathway.