Neural stem cells (NSCs) in the spinal cord hold great potential for repair after spinal cord injury (SCI). The ependyma in the central canal (CC) region has been considered as the NSCs source in the spinal cord. However, the ependyma function as NSCs after SCI is still under debate. We used Nestin as a marker to isolate potential NSCs and their immediate progeny, and characterized the cells before and after SCI by single-cell RNA-sequencing (scRNA-seq). We identified two subgroups of NSCs: the subgroup located within the CC cannot prime to active NSCs after SCI, while the subgroup located outside the CC were activated and exhibited the active NSCs properties after SCI. We demonstrated the comprehensive dynamic transcriptome of NSCs from quiescent to active NSCs after SCI. This study reveals that Nestin+ cells outside CC were NSCs that activated upon SCI and may thus serve as endogenous NSCs for regenerative treatment of SCI in the future.Keywords: Nestin; Smart-seq2; ependymal cell; neural stem cell; scRNA-seq.