The objective of this study was to investigate the mechanism of the soluble Aβ oligomer-induced alteration in synaptic proteins. Therefore, an immunofluorescence technique was applied to investigate the changes in the expression of postsynaptic density-95 (PSD-95) protein in primary hippocampal neurons, which was exposed to Aβ25–35 after N-methyl-D-aspartate receptor (NMDAR) antagonist or agonist treatment. Results showed that Aβ25–35 downregulated PSD-95 protein in a dose- and time-dependent manner. Treatment of cells with MK-801 (a general NMDA receptor antagonist) prevented Aβ-induced PSD-95 degradation. Moreover, when extrasynaptic NMDA receptors were blocked by ifenprodil (a specific antagonist of the NR2B subunit), the Aβ-induced downregulation of PSD-95 was significantly attenuated. However, when synaptic NMDA receptors were blocked by bicuculline (a GABA receptor antagonist) in combination with MK-801, the PSD-95 degradation did not change significantly. The results suggest that Aβ-induced downregulation of PSD-95 depends on NMDAR activity, and extrasynaptic NMDA receptors may be involved in Aβ-induced synaptic protein degradation.