Excitotoxicity resulting from over-stimulation of glutamate receptors is a major cause of neuronal death in cerebral ischemic stroke. The over-stimulated ionotropic glutamate receptors exert their neurotoxic effects by over-activation of calpains which induce neuronal death by catalysing limited proteolysis of specific cellular proteins. Here, we report that in cultured cortical neurons and in vivo in a rat model of focal ischemic stroke, the tyrosine kinase Src is cleaved by calpain at a site in the N-terminal unique domain. This generates a truncated Src fragment of approximately 52 kDa, which we localized predominantly to the cytosol. A cell membrane-permeable fusion peptide derived from the unique domain of Src effectively prevents calpain from cleaving Src in neurons and protects against neuronal death induced by glutamate over-stimulation. To explore the role of the truncated Src fragment in neuronal death, we studied the effect of lentivirus-directed expression of a recombinant truncated Src fragment on survival of cultured neurons. Expression of this fragment, which lacks the myristoylation motif and unique domain, was sufficient to induce neuronal death. Furthermore, the kinase activity is indispensable to its neurotoxic action. As Src maintains neuronal survival, our results implicate calpain cleavage as a molecular switch converting Src from a promoter of cell survival to a mediator of neuronal death in excitotoxicity. Besides unveiling a new function of Src, our discovery of the neurotoxic action of the truncated Src fragment suggests new therapeutic strategies with potential to minimize brain damage in ischemic stroke.