To examine the effects of HGF on synaptic densities under excitotoxic conditions, we investigated changes in the number of puncta detected by double immunostaining with NMDA receptor subunits and presynaptic markers in cultured hippocampal neurons. Exposure of hippocampal neurons to excitotoxic NMDA (100 μM) decreased the synaptic localization of NMDA receptor subunit NR2B, whereas synaptic NR1 and NR2A clusters were not altered. Colocalization of PSD-95, a scaffolding protein of the receptor, with the presynaptic protein synapsin I was also decreased after excitotoxicity. Treatment with HGF attenuated these decreases in number. The decrease in the levels of surface NR2B subunits following the addition of the excitotoxic NMDA was also attenuated by the HGF treatment. The decrease in CREB phosphorylation in response to depolarization-evoked NMDA receptor activation was prevented by the HGF treatment. These results suggest that HGF not only prevented neuronal cell death but also attenuated the decrease in synaptic localization of NMDA receptor subunits and prevented intracellular signaling through the NMDA receptor.