During brain ischemia, glutamate has excitotoxic effects to neurons through excessive activation of its receptor. Na+/H+ exchangers-1 (NHE-1) activity has been known to play a critical role in neuronal injury during glutamate excitotoxicity. It has been recently reported that inhibition of NHE-1 during ischemia/reperfusion injury has protective effects in brain. However, the protective mechanism of NHE-1 inhibition has not been clearly elucidated during glutamate excitotoxicity. In the present study, inhibition of NHE-1 with the potent inhibitors, cariporide and KR-33028 reduced glutamate-induced apoptotic and necrotic cell death in cortical neuronal cells. During glutamate exposure, dual peaks of cytosolic and mitochondrial Ca2+ rise were observed, and NHE-1 inhibitors did not suppress the initial peak whereas reduced the secondary peak. NHE-1 inhibitors also inhibited dissipation of mitochondrial membrane potential, cytochrome c release and ROS accumulation following glutamate exposure, suggesting its inhibitory effect on glutamate-mediated mitochondrial death pathways. In this study, I further demonstrated that the NHE-1 activity following glutamate was regulated by protein kinase C-β (PKC-β) in neuronal cells. The activation of PKC-β was significantly increased during glutamate stimulation, and NHE-1 activity and its phosphorylation during glutamate exposure was attenuated by the PKC-β inhibition with Go6976. I also examined the extracellular signal-related kinases (ERK)-p90kDa ribosomal S6 kinase (p90RSK) signaling pathway. At glutamate exposure, phosphorylated NHE-1 was increased with a concurrent elevation of phosphorylation of ERK and p90RSK. Inhibition of ERK and p90RSK activity with U0126 and SL0101 abolished activity and phosphorylation of NHE-1, respectively. Moreover, neuroprotection was observed with Go6976, U0126 and SL0101 following glutamate stimulation, respectively. Taken together, these results suggest that activation of PKC-β-ERK-p90RSK pathways following glutamate phosphorylates NHE-1 and increases its activity, which subsequently contributes to neuronal apoptotic and necrotic cell death via mitochondrial death pathway. I conclude that targeted inhibition of PKC-β-ERK-p90RSK pathway and reduction of NHE-1 activity in response to agonists such as glutamate is a novel strategy to brain ischemic injury.