Objective To explore the role and possible regulatory mechanism of hippocampal N-methyl-D-aspartate receptor subtype 3A（GluN3A） subunit in anxiety-related behaviors，ability of learning and memory，and depression-like behaviors. Methods Thirty male C57BL/6N mice aged 7-8 weeks were randomly divided into GluN3A overexpression group（GluN3A OE） and control group. Recombinant adeno-associated viruses with（rAAV-GluN3A） and without（rAAV-control） the gene encoding GluN3A were injected into the bilateral hippocampus of mice in the GluN3A OE and control groups，respectively. At 3 weeks after virus injection，behavioral assessments were conducted using the open field test，Y maze test，forced swimming test，and tail suspension test. Subsequently，Western blot and qRT-PCR were performed to determine the expression levels of GluN3A，myelin basic protein（MBP），myelin-associated glycoprotein（MAG），and oligodendrocyte transcription factor 2（Olig2）. Moreover，immunofluorescence staining was carried out to evaluate the development of oligodendrocytes. Results In the open field test，there were no significant differences in the total moving distance（P=0.256） and the time spending in central area of open field（P=0.487） between the two groups. In the Y maze test，the total number of arm entries of mice was significantly lower in the GluN3A OE group than in the control group（P=0.015），while no significant difference was observed in alternating rate between the two groups（P=0.381）. Both forced swimming test（P=0.347） and tail suspension test（P=0.471） showed no significant differences in immobility time between the two groups. After injection of rAAV-GluN3A in the bilateral hippocampus，the expression of GluN3A protein in the hippocampus was significantly increased compared to that of control group（P=0.005）. The expression levels of MBP protein（P=0.019），MAG protein（P=0.022），and Olig2 protein（P=0.022） were significantly decreased in mice of the GluN3A OE group compared to mice of the control group. The expression levels of MBP mRNA（P=0.043），MAG mRNA（P=0.032），and Olig2 mRNA（P<0.001） were significantly decreased in mice of the GluN3A OE group，which was consistent with the decreased expression of MBP，MAG，and Olig2 proteins. Immunofluorescence showed that the density of CC1⁺ mature oligodendrocytes was significantly decreased in the hippocampus of mice in the GluN3A OE group（P<0.001），while the density of PDGFRɑ⁺ oligodendrocyte precursor cells displayed negligible variation（P=0.542）. Conclusion Overexpression of GluN3A subunit gene in the bilateral hippocampus did not lead to changes in ability of learning and memory，anxious behaviors，and depression-like behaviors in mice，but led to the low expression of myelin-related proteins and decreased density of mature oligodendrocytes in the hippocampus.