Transient receptor potential melastatin-4 (TRPM4) forms a complex with N-methyl-D-aspartate receptors (NMDARs) that facilitates NMDAR-mediated neurotoxicity. Here we used pharmacological tools to determine how TRPM4 regulates NMDAR signaling. Brophenexin, a compound that binds to TRPM4 at the NMDAR binding interface, protected hippocampal neurons in culture from NMDA-induced death, consistent with published work. Brophenexin (10 μM) reduced NMDA-evoked whole-cell currents recorded at 22°C by 87% ± 14% with intracellular Ca(2+) chelated to prevent TRPM4 activation. Brophenexin inhibited NMDA-evoked currents recorded in Na(+)-free solution by 87% ± 13%, suggesting that brophenexin and TRPM4 modulate NMDAR function. Incubating cultures in Mg(2+)-free buffer containing tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione, and bicuculline for 30 min inhibited NMDA-evoked increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) recorded at 22°C by 50% ± 18% and prevented inhibition by brophenexin. In the absence of these inhibitors, brophenexin inhibited the NMDA-evoked response by 51% ± 16%. Treatment with the TRPM4 inhibitor 4-chloro-2-(1-naphthyloxyacetamido)benzoic acid (NBA; 10 μM) increased NMDA-evoked Ca(2+) influx by 90% ± 15%. Increasing extracellular NaCl to 237 mM, a treatment that activates TRPM4, inhibited the NMDA-evoked increase in [Ca(2+)](i) by a process that occluded the inhibition produced by brophenexin and was prevented by NBA. In recordings performed at 32°C-34°C, brophenexin inhibited the NMDA-evoked [Ca(2+)](i) response by 42% ± 10% but NBA was without effect. These results are consistent with a model in which TRPM4 interacts with NMDARs to potentiate Ca(2+) flux through the NMDAR ion channel and thus provides a potential mechanism for the neuroprotection afforded by NMDAR/TRPM4 interface inhibitors such as brophenexin.