Background: Thousands of infants and children are undergoing anesthesia around the world every day. But impacts of anesthetics on the developing neural system remain unclear yet. Previous evidence showed that anesthesia might affect the developing neural system. Thus, early-life anesthesia becomes a critical issue in clinical pediatric practice. Hence, propofol, a short-acting and widely applied intravenous anesthetic, has been gaining focus upon neonatal anesthesia. Methods: Fifty-four male C57BL/6J mice were randomly divided into following three groups: group D6 intraperitoneally (i.p.) injected propofol (100mg/kg body weight) once a day from postnatal day 6 (P6) to P11, group D1 administrated propofol (100mg/kg, i.p.) at P6 solely and administrated normal saline (10ml/kg, i.p.) from P7 to P11, and group N treated with normal saline (10ml/kg, i.p.) from P6 to P11 as the control (n=18 per group). Then, at P28, nine mice were collected randomly from each group for NR2B membrane translocation and phosphorylation analysis, and the rest half in each group were assigned to perform Morris water maze tests from P28 to P35. Results: Results showed that total protein expression levels of NR2B increased (p<.001) while its membrane translocation decreased (p<.001, n=9 per group) in the hippocampus but not in the prefrontal cortex of neonatal mice after repeated propofol administration. Phosphorylation levels of NR2B at serine 1303 (D1: p<.05; D6: p<.001, n=9 per group) and serine 1480 (D1: p<.01, D6: p<.001, n=9 per group) increased significantly as well in the hippocampus compared with group N. In addition, memory deficits (p<.05, n=9 per group) were observed in Morris water maze tests of group D6 mice. Conclusions: These results suggested that propofol exposure downregulates NR2B membrane translocation and causes spatial memory deficits, with a mediated increased NR2B protein expression and phosphorylation at Ser1303/1480 residues in the hippocampus of neonatal mice.