The mental retardation-associated protein, srGAP3 is highly expressed in neurogenic sites. It is thought to regulate the key aspects of neuronal development and functions. Little is known about the interaction between srGAP3 and immature neural stem cells/neural progenitor cells (NSCs/NPCs). In the current study, the expression of srGAP3 in NSCs/NPCs was detected. Then, survival, proliferation, differentiation, and morphological alteration of NSCs/NPCs were assessed after a lentivirus-mediated knockdown of srGAP3. The results showed that srGAP3 is highly expressed in NSCs/NPCs both in vitro and in vivo. After knockdown of srGAP3 (LV3-srGAP3 infection), viability and proliferation of NSCs/NPCs dramatically decreased, approximately 85% displayed a similar morphology with type I cells that have no or only few indistinguishable processes. After 7 days culture in a differentiation medium, 62.5%±8.3% of cells in the srGAP3 knockdown group were nestin-positive and 24.8%±5.8% of them were β-tubulin III-positive, which are significantly higher (30.2%±9.9% and 14.6%±2.7%) than in the control group (LV3-NC infection). In addition, cells in the knockdown group had significantly fewer, but longer processes. Our results demonstrate that srGAP3 knockdown negatively regulates NSCs/NPCs survival, proliferation, differentiation, and morphological alteration, particularly, process formation. Taken together, our results provide strong evidence that srGAP3 is involved in the regulation of biological behavior and the morphological features in rat NSCs/NPCs in vitro.