CDK5 activation promotes ischemic neuronal death in stroke, with the recognized activation mechanism being calpain-dependent p35 cleavage to p25. Here we reported that CDK5-Tyr15 phosphorylation by zinc induced CDK5 activation in brain ischemic injury. CDK5 activation and CDK5-Tyr15 phosphorylation were observed in the hippocampus of the rats that had been subjected to middle cerebral artery occlusion, both of which were reversed by pretreatment with zinc chelator; while p35 cleavage and calpain activation in ischemia were not reversed. Zinc incubation resulted in CDK5-Tyr15 phosphorylation and CDK5 activation, without increasing p35 cleavage in cultured cells. Site mutation experiment confirmed that zinc-induced CDK5 activation was dependent on Tyr15 phosphorylation. Further exploration showed that Src kinase contributed to zinc-induced Tyr15 phosphorylation and CDK5 activation. Src kinase inhibition or expression of an unphosphorylable mutant Y15F-CDK5 abolished Tyr15 phosphorylation, prevented CDK5 activation and protected hippocampal neurons from ischemic insult in rats. We conclude that zinc-induced CDK5-Tyr15 phosphorylation underlies CDK5 activation and promotes ischemic neuronal death in stroke.