BACKGROUND Dexmedetomidine (Dex) is a highly selective α2-adrenoceptor agonist used as an off-label medication for pediatric sedation and analgesia. Recently, Dex was reported to exhibit neuroprotective efficacy in several brain injury models. Here we investigate whether neonatal Dex administration promotes hippocampal neurogenesis and enhances hippocampus-dependent spatial learning and memory under physiological conditions. METHODS Postnatal day 7 (P7) pups were administered saline (vehicle control) or Dex (10, 20, or 40 µg/kg) by intraperitoneal injection. Neurogenesis and astrogenesis were examined in brain slices by BrdU immunostaining on P8 and changes in the expression levels of GDNF, NCAM, CREB, PSD95, and GAP43 were assessed by Western blotting on P35, respectively. Open field and Morris water maze (MWM) tests were conducted from P28 to P36 in order to assess effects on general motor activity and spatial learning, respectively. RESULTS Dexmedetomidine at 20 µg/kg significantly enhanced neurogenesis and astrogenesis in hippocampus and upregulated GDNF, NCAM, CREB, PSD95, and GAP43 compared to vehicle and other Dex doses. Moreover, 20 µg/kg Dex-injected rats showed no changes in motor or anxiety-like behavior but performed better in the MWM test compared to all other groups. CONCLUSIONS Neonatal injection of Dex (20 µg/kg) enhances spatial learning and memory in rat pups, potentially by promoting hippocampal neurogenesis and synaptic plasticity via activation of GDNF/NCAM/CREB signaling.