We aimed to investigate whether ethanol (EtOH) and acetaldehyde (AcH) can affect glutamate and its receptors GluN1 and GluA1 in the hippocampus of Aldh2-knockout (Aldh2-KO) and C57BL/6N (wild-type (WT)) mice. To do this, we first examined the effect of local administration of EtOH (100 mM, 200 mM, and 500 mM) and AcH (100 μM, 200 μM, and 500 μM) on extracellular glutamate levels in freely moving mice. Retrodialysis of 200 mM and 500 mM EtOH into the hippocampus of WT and Aldh2-KO mice produced significant decreases in extracellular glutamate levels (p < 0.05). A dose of 500 mM EtOH induced a greater decrease in Aldh2-KO mice (p < 0.05) than in WT mice, indicating the action of AcH. Similarly, perfusion of 200 μM and 500 μM AcH decreased glutamate in Aldh2-KO mice (p < 0.05), but this decrease was not seen in WT mice at any AcH dose. Second, we tested whether the EtOH- and AcH-induced decrease in glutamate was associated with decreases in GluN1 and GluA1 expression, as measured by real-time PCR and Western blot. We found a significant decrease in GluN1 (p < 0.05) and GluA1 (p < 0.05) subunits after a high dose of EtOH (4.0 g/kg) and AcH (200 mg/kg) in WT mice. However, a 2.0 g/kg dose of EtOH did not produce a consistent decrease in GluN1 or GluA1 between messenger RNA and protein. In Aldh2-KO mice, all three doses of EtOH (1.0 g/kg, 2.0 g/kg, and 4.0 g/kg) and AcH (50 mg/kg, 100 mg/kg, and 200 mg/kg) decreased GluN1 expression (p < 0.05), while moderate-to-high doses of EtOH (2.0 g/kg and 4.0 g/kg) and AcH (100 mg/kg and 200 mg/kg) decreased GluA1 expression (p < 0.05). Together, these in vivo and ex vivo data suggest that EtOH and AcH decrease extracellular glutamate in the hippocampus of mice with a concomitant decrease in GluN1 and GluA1 subunits, but these effects require relatively high concentrations and may, therefore, explain the consequences of EtOH intoxication.