Ischemic neuronal damage is induced by various factors such as glutamate excitotoxicity and oxidative stress. Excessive amount of extracellular glutamate followed by cerebral ischemia is a major factor to lead intracellular Ca²⁺ overload through the N-methyl-D-aspartate (NMDA) receptor and then causes neuronal death. It has been known that several proteolytic enzymes are stimulated by intracellular Ca²⁺ elevation. However, roles of proteolytic enzymes on NMDA-induced neuronal damage are not fully defined. We examined whether inhibitions of proteolytic enzymes protected primary cultured rat cortical neurons from NMDA-induced damage. Among several inhibitors, furin inhibitor markedly protected neurons from NMDA-induced damage in a dose-dependent manner. Furthermore, calpain activation led by NMDA treatment was inhibited by the furin inhibitor. We next investigated the ability of furin inhibitor to attenuate cell damage in a rat cerebral ischemia model. We demonstrated that infarct volume after cerebral ischemia was not affected by treatment with furin inhibitor. Although further research will be needed to assess the therapeutic potential of furin inhibitor in cerebral ischemia in vivo, this study revealed a novel role of furin in excitotoxic injury in cortical neurons.