Increasing evidence indicates that the Eph receptors and their ephrin ligands are involved in the regulation of interactions between neurons and astrocytes. Moreover, astrocytic ephrin-A3 reverse signaling mediated by EphA4 receptors is necessary for controlling the abundance of glial glutamate transporters. However, the role of ephrin-A3 reverse signaling in astrocytic function and neuronal death under ischemic conditions remains unclear. In the present study, we found that the EphA4 receptor and its ephrin-A3 ligand, which were distributed in neurons and astrocytes, respectively, in the hippocampus showed a coincident up-regulation of protein expression in the early stage of ischemia. Application of clustered EphA4 decreased the expressions of astrocytic glutamate transporters together with astrocytic glutamate uptake capacity through activating ephrin-A3 reverse signaling. In consequence, neuronal loss was aggravated in the CA1 region of the hippocampus accompanied by impaired hippocampus-dependent spatial memory when clustered EphA4 treatment was administered prior to transient global ischemia. These findings indicate that EphA4-mediated ephrin-A3 reverse signaling is a crucial mechanism for astrocytes to control glial glutamate transporters and prevent glutamate excitotoxicity under pathological conditions.