AbstractExcessive N-methyl-D-aspartate receptor (NMDAR) activation and the resulting activation of neuronal nitric oxide synthase (nNOS) cause neuronal injury. Homer1b/c facilitates NMDAR-PSD95-nNOS complex interactions, and Homer1a is a negative competitor of Homer1b/c. We report that Homer1a was both upregulated by and protected against NMDA-induced neuronal injury in vitro and in vivo. The neuroprotective activity of Homer1a was associated with NMDA-induced Ca2+ influx, oxidative stress and the resultant downstream signaling activation. Additionally, we found that Homer1a functionally regulated NMDAR channel properties in neurons, but did not regulate recombinant NR1/NR2B receptors in HEK293 cells. Furthermore, we found that Homer1a detached the physical links among NR2B, PSD95 and nNOS and reduced the membrane distribution of NMDAR. NMDA-induced neuronal injury was more severe in Homer1a homozygous knockout mice (KO, Homer1a−/−) when compared with NMDA-induced neuronal injury in wild-type mice (WT, Homer1a+/+). Additionally, Homer1a overexpression in the cortex of Homer1a−/− mice alleviated NMDA-induced neuronal injury. These findings suggest that Homer1a may be a key neuroprotective endogenous molecule that protects against NMDA-induced neuronal injury by disassembling NR2B-PSD95-nNOS complexes and reducing the membrane distribution of NMDARs.