Objective To analyze proliferation and differentiation of glial fibrillary acid protein (GFAP)- and nestin-positive (GFAP+/nestin+) cells isolated from the subventricular zone following fluid percussion brain injury to determine whether GFAP+/nestin+ cells exhibit characteristics of neural stem cells. Methods Male Sprague-Dawley rats, aged 12 weeks and weighing 200-250 g, were randomly and evenly assigned to normal control group and model group. In the model group, a rat model of fluid percussion brain injury was established. Five days later, subventricular zone tissue was resected from each group and made into single cell suspension. After serum-free neural stem cell medium culture and subsequent serum-induced differentiation, cell type, proliferation and differentiation capacities were determined by immunofluorescence staining and flow cytometry. Results At 3-7 days after fluid percussion brain injury, nestin+/GFAP+ cells in the single cell suspension from the model group significantly outnumbered those from the normal control group (P<0.01). In the model group, an increased number of small neurospheres with smooth cell edge and bulged center formed after primary culture, and were clearly visible with the increase of culture time and medium replacement. After several passages, many clonal spheres were obtained, suggesting strong self-proliferatiing capacity. Neurospheres from the model group differentiated into astrocytes, neurons and oligodendrocytes. Conclusion GFAP+/nestin+ cells isolated from the adult rat subventricular zone after fluid percussion brain injury are thought to be neural stem cells because of their self-renewal and multi-differentiation capacities.