Neu2000 [NEU, 2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoic acid], a derivative of sulfasalazine, attenuates NMDA-induced neuronal toxicity. Here we investigated the effects of NEU on the NMDA receptor (NMDAR) using whole-cell patch clamp technique to determine the molecular mechanisms underlying its neuroprotective role. NEU reversibly suppressed NMDA responses in an uncompetitive manner with fast binding kinetics. Its inhibition of NMDAR activity depended on both the concentration and the use of agonist but not on the membrane potential. NEU accelerated NMDA desensitization without affecting the binding affinity of NMDAR for its agonists and stabilized the closed state of NMDAR. Therefore, NEU should effectively alleviate disorders that are a result of glutamate excitoxicity with fewer side effects because it is a low-affinity gating modifier that antagonizes NMDAR in an uncompetitive manner. Moreover, in the presence of ifenprodil (an NR2B antagonist) but not NVP-AAM077 [(R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydro-quinoxalin-5-yl)-methyl]-phosphonic acid, an NR2A antagonist], the extent of NEU block was decreased, suggesting that NEU is an NR2B-specific antagonist.