We hypothesize that stimulation of Na+–K+–Cl+ cotransporter (NKCC1) causes Na+ overload that may lead to reversal of Na+–Ca2+ exchanger isoform 1 (NCX1) and ischemic neuronal damage. NCX1 protein expression and Ca2+ influx via reversal of NCX were decreased by ∼70% in NCX1+/− neurons. Compared to NCX1+/+ neurons, NCX1+/− neurons exhibited significantly less cell death (∼30%) after 3 h oxygen and glucose deprivation (OGD) and 21 h reoxygenation. Additional neuroprotection was found in NCX1+/− neurons treated with NCX inhibitor KB-R7943. Moreover, expression of NCX1 protein was ∼40% lower in NCX1+/− brains than in NCX1+/+ brains. However, there was no significant reduction in cerebral infarction in NCX1+/− mice following middle cerebral artery occlusion (MCAO). These data suggest that moderate reduction of NCX1 protein may be not enough to exert protection. We used small RNA-interference (siRNA) approach to further elucidate the role of NCX1 in ischemic cell damage. Efficacy of anti-NCX1 siRNA was tested in astrocytes and ∼50% knockdown of NCX1 protein expression was achieved after 24–72 h transfection. Reduction in NCX1 protein expression was also found in brains of NCX1+/− mice after the siRNA injection. NCX1+/− mice treated with siRNA showed ∼20% less MCAO-induced infarction, compared to NCX1+/− mice. Approximately 50% neuroprotection was detected in NKCC1+/−/NCX1+/− mice following MCAO. In conclusion, these data suggest that NCX1 plays an important role in ischemia/reperfusion-induced neuronal injury.