Neuronal excitotoxicity induced by spreading depolarization occurs during multiple brain diseases. The subsequent extensive releasing of neuronal transmitter glutamate results in over activation of the ionic glutamate receptors and then triggers neuronal cell death. The N-methyl-D-aspartate (NMDA) receptor is one major type of excitatory ionic glutamate receptors in the central nervous system, and it exerts vital functions on the membrane of neurons. Distinct subtypes of the NMDA receptor play different roles and their expression was dynamically regulated according to both physiological and pathological stimulations. During neuronal excitotoxicity the expression of the GluN2A-containing NMDA receptor is specifically up-regulated, and as a result, the ratio of GluN2A- versus GluN2B-containing NMDA receptors is altered. However the physiological significance of this phenomenon is still not clear. In this research, we specifically inhibited the increase of the GluN2A-containing NMDA receptor by a peptide without affecting the basic expression of both GluN2A- and GluN2B-containing NMDA receptors, and found that the oxidative stress of neurons was intensified, with increased endogenous reactive oxygen species (ROS), loss of mitochondrial membrane potential, and elevated expressions of Bcl-2-associated X protein (Bax) and apoptosis-inducing factor (AIF). Furthermore, the phosphorylation of Akt and ERK were also inhibited. These results indicated that the dynamic expression of the GluN2A-containing NMDA receptor played crucial roles in protecting neurons from excitotoxicity.