SignificanceGlutamate is the principal excitatory neurotransmitter in the brain. Kainate receptors, a subfamily of the ionotropic glutamate receptors, mediate the pre- and postsynaptic actions of glutamate. Overactivation of postsynaptic kainate receptors plays an important role in neurodegeneration after ischemic stroke. Because Src kinases show increased activity after brain ischemia, we evaluated their roles in regulating kainate receptor function after brain ischemia. Our results demonstrate that Src kinases bind to and phosphorylate GluK2 at Y590 and facilitate kainate-evoked whole-cell currents and calcium influx. Furthermore, long-term kainate stimulation promotes apoptosis through the endocytosis of GluK2 subunits and activation of JNK3–c-Jun signaling. In summary, our results demonstrate that Src phosphorylation of GluK2 regulates kainate receptor activity and downstream excitatory signaling.