Recent years, the number of neonatal patients receiving surgery under general anesthesia is increasing. Previous studies have indicated that ketamine can disturb the proliferation and differentiation of developing neural stem cells (NSCs). Therefore, the safe use of ketamine in pediatric anesthesia has drawn great concern among anesthesiologists and children’s parents. Dexmedetomidine (DEX) is widely used in sedation, antianxiety and analgesia. Recent studies have shown that DEX could provide neuroprotection against anesthetic-induced neurotoxicity in the developing brain. The aim of this in vivo study was to investigate whether DEX had neuroprotective effects on the proliferation and differentiation of NSCs in the subventricular zone (SVZ) following neonatal ketamine exposure. Methods: Postnatal day 7 (PND-7) male Sprague-Dawley rats were equally divided into the following 5 groups: Control group (n=8), ketamine group (n=8), 1 μg/kg DEX+ketamine group (n=8), 5 μg/kg DEX+ketamine group (n=8) and 10 μg/kg DEX+ketamine group (n=8). The proliferation and differentiation of NSCs in the SVZ were assessed by immunostaining with BrdU incorporation. Results: Neonatal ketamine exposure significantly inhibited NSC proliferation and astrocytic differentiation in the SVZ, and neuronal differentiation was markedly promoted. Furthermore, DEX pretreatment reversed the ketamine-induced disturbances in the proliferation and differentiation of NSCs at moderate (5 μg/kg) or high doses (10 μg/kg). Conclusion: Our findings demonstrate that DEX may have neuroprotective effects on NSCs in the SVZ of neonatal rats in a repeated ketamine anesthesia model.