Objective: The proliferation and differentiation of developing neural stem cells (NSCs) have been particularly interesting targets to study ketamine-induced neurotoxicity. Our previous findings have shown that neonatal ketamine exposure inhibits the proliferation of NSCs in the hippocampal dentate gyrus (DG) and promotes neuronal differentiation. However, the potential mechanisms are poorly understood. Notch signalling pathway plays an important role in the regulation of neurogenesis. The objective of this study was to investigate whether Notch signalling pathway was involved in neurogenesis impairment and long-term neurocognitive dysfunction caused by neonatal ketamine exposure. Methods: Postnatal day 7 (PND-7) male Sprague-Dawley (SD) rats were intraperitoneally injected with normal saline (NS) or 40 mg/kg ketamine for four consecutive times (40 mg/kg×4) at an interval of 1 h. Notch ligand Jagged1 (0.5 mg/kg) was micro-injected into the hippocampal DG with the stereotactic apparatus at 1 h before NS or ketamine administration. Lentivirus over-expressing Notch1 intracellular domain (LV-NICD1) was micro-injected into the hippocampal DG 4 days before NS or ketamine administration. Western blot was used to detect the changes of Notch1 signalling pathway related proteins in the hippocampal DG at 24 h after administration. The S-phase marker 5-bromodeoxyuridine (BrdU) was administered immediately after the treatment, then the proliferation and differentiation of NSCs in hippocampal DG were detected by using double-immunofluorescence staining at 24 h after treatment. Moreover, the changes of hippocampus-dependent spatial memory in the adult rats were tested by Morris water maze test in 2-month-old rats. Results: Ketamine anesthesia in neonatal rats decreased the expression levels of Jagged1, Notch1, Notch1 intracellular domain (NICD1) and hairy enhancer of split 1 (Hes1), and inhibited the proliferation and astrocytic differentiation of NSCs and promoted neuronal differentiation. Neonatal ketamine exposure induced the deficit in hippocampus-dependent spatial reference memory tasks in 2-month-old rats. The micro-injection of Jagged1 or LV-NICD1 reversed the inhibitory effect of ketamine on the expression of Notch1 related proteins in the hippocampal DG, and attenuated the ketamine-induced interference of NSCs proliferation and differentiation. In addition, Morris water maze test suggested that the administration of Jagged1 or LV-NICD1 could improve cognitive function in 2-month-old rats after neonatal ketamine exposure. Conclusions: These results suggest that neonatal exposure to ketamine in rats interferes with the proliferation and differentiation of hippocampal NSCs and impairs neurocognitive function in adulthood via inhibition of Notch1 signalling pathway. These findings contribute to further understanding of the neonatal neurotoxicity induced by ketamine and its underlying mechanisms.