Background α-Synuclein misfolding and aggregation contribute to synaptic dysfunction in synucleinopathies, including Parkinson’s disease. However, the mechanism underlying the effect of α-synuclein on synaptic components remains unclear. Since the N-methyl-D-aspartic acid receptor (NMDAR) plays a key role in glutamate synapse pathophysiology, we here investigated its surface dynamics and functional distribution in neurons exposed to various pathological α-synuclein forms. Methods A combination of single-molecule tracking, immunochemistry, immunoblot and calcium imaging approaches were used to assess the changes in NMDAR membrane dynamics and functions. The NMDAR alterations were evaluated in rat cultured hippocampal networks, in which α-synuclein mutants were overexpressed or exposed to α-synuclein proteins (monomeric/PFF α-synuclein). The surface dynamics of NMDAR subtype was artificially tuned in order to test its instrumental role. Results We observed that mutant α-synuclein (A53T-α-synuclein) restricted NMDAR surface trafficking and impaired synaptic function. In contrast, wild-type α-synuclein did not affect synaptic NMDAR. Further, we found that chronic exposure to α-synuclein preformed fibrils induced molecular dysfunctions that mainly targeted the GluN2B-NMDAR subtype. The deficits of synaptic NMDAR have also been found in A53T transgenic mice α-synuclein. Upon fine-tuning of the surface dynamics of GluN2B-NMDAR, pathological α-synuclein gradually lost its synaptic toxicity. Conclusions Our findings indicate that pathological α-synuclein alters GluN2B-NMDAR synaptic dynamics and organization, which leads to glutamate synapse dysfunction.