Neural excitotoxicity is a typical factor in the early phase pathogenesis of cerebral ischemia. Its cellular and molecular mechanisms are still unclear and clinical approaches are still lacking of promising therapies. We have examined the vulnerability of cortical neurons to short-term ischemia in rats by simultaneously analyzing the activities of inhibitory and principal neurons in brain slices. Our results demonstrate that short-term in vitro ischemia permanently impairs the excitability of inhibitory neurons (IN) and synaptic transmission mediated by gamma-aminobutyric acid (GABA). However, principal neurons appear to be more exciting during the reperfusion. The vulnerability of inhibitory neurons to ischemia acquires during postnatal development. Our findings signify a major contribution of the ischemic dysfunction of inhibitory neurons to neural excitotoxicity as well as a strategy to prevent the progress of ischemic stroke by protecting inhibitory neurons.