Activation of protein kinase C{small element of} (PKC{small element of}) confers protection against neuronal ischemia/reperfusion. Activation of PKC{small element of} leads to its translocation to multiple intracellular sites, so a mitochondria-selective PKC{small element of} activator was used to test the importance of mitochondrial activation to the neuroprotective effect of PKC{small element of}. PKC{small element of} can regulate key cytoprotective mitochondrial functions, including electron transport chain activity, reactive oxygen species (ROS) generation, mitochondrial permeability transition, and detoxification of reactive aldehydes. We tested the ability of mitochondria-selective activation of PKC{small element of} to protect primary brain cell cultures or mice subjected to ischemic stroke. Pretreatment with either general PKC{small element of} activator peptide, TAT-Ψ{small element of}RACK, or mitochondrial-selective PKC{small element of} activator, TAT-Ψ{small element of}HSP90, reduced cell death induced by simulated ischemia/reperfusion in neurons, astrocytes, and mixed neuronal cultures. The protective effects of both TAT-Ψ{small element of}RACK and TAT-Ψ{small element of}HSP90 were blocked by the PKC{small element of} antagonist {small element of}V1-2, indicating that protection requires PKC{small element of} interaction with its anchoring protein, TAT-{small element of}RACK. Further supporting a mitochondrial mechanism for PKC{small element of}, neuroprotection by TAT-Ψ{small element of}HSP90 was associated with a marked delay in mitochondrial membrane depolarization and significantly attenuated ROS generation during ischemia. Importantly, TAT-Ψ{small element of}HSP90 reduced infarct size and reduced neurological deficit in C57/BL6 mice subjected to middle cerebral artery occlusion and 24 hr of reperfusion. Thus selective activation of mitochondrial PKC{small element of} preserves mitochondrial function in vitro and improves outcome in vivo, suggesting potential therapeutic value clinically when brain ischemia is anticipated, including neurosurgery and cardiac surgery. © 2013 Wiley Periodicals, Inc.