Midazolam, a widely used anesthetic, inhibits proliferation of neural stem cells (NSCs) and induces neuroapoptosis in neonates. Dexmedetomidine, an effective auxiliary medicine in clinical anesthesia, protects the developing brain against volatile anesthetic-induced neuroapoptosis. Whether dexmedetomidine protects against neurogenesis damage induced by midazolam remains unknown. This study aims to clarify the protective effect of dexmedetomidine on midazolam-induced neurogenesis damage and explore its potential mechanism. Postnatal 7-day-old Sprague–Dawley (SD) rats and cultured NSCs were treated with either normal saline, midazolam, or dexmedetomidine combined with midazolam. The rats were sacrificed at 1, 3, and 7 days after treatment. Cell proliferation was assessed by 5-bromodeoxyurdine (BrdU) incorporation. Cell viability was determined using MTT assay. Cell differentiation and apoptosis were detected by immunofluorescent staining and terminal dUTP nick-end labeling (TUNEL), respectively. The protein levels of p-JNK, p-P38, and cleaved caspase-3 were quantified using Western blotting. Midazolam decreased cell proliferation and increased cell apoptosis in the subventricular zone (SVZ), the subgranular zone (SGZ) of the hippocampus, and cultured NSCs. Moreover, midazolam decreased cell viability and increased the expression of p-JNK and p-P38 in cultured NSCs. Co-treatment with dexmedetomidine attenuated midazolam-induced changes in cell proliferation, viability, apoptosis, and protein expression of p-JNK and p-P38 in cultured NSCs. Midazolam and dexmedetomidine did not affect the differentiation of the cultured NSCs. These results indicate that dexmedetomidine alleviated midazolam-induced neurogenesis damage via JNK and P38 MAPK pathways.