The transcription factor NF-E2-related factor 2 (Nrf2) mediates transcription of antioxidant/cytoprotective genes by binding to the antioxidant-response element (ARE) within DNA. Upregulation of these genes constitutes a pleiotropic cytoprotective defense pathway, which has been shown to produce neuroprotection in numerous models by decreasing lipid peroxidation (LP) as measured by the neurotoxic LP by-product 4-hydroxynonenal (4-HNE). As neuronal mitochondria have previously been shown to be susceptible to insult-induced LP-mediated oxidative damage, we sought to mechanistically investigate whether Nrf2–ARE activation in vivo could protect mitochondria from subsequent 4-HNE exposure ex vivo. Young adult male CF-1 mice were administered one of two known Nrf2–ARE activators as single intraperitoneal doses—sulforaphane (SFP; 5.0 mg/kg) or carnosic acid (CA; 1.0 mg/kg)—or their respective vehicles 48 h before Ficoll isolation of rat cerebral cortical mitochondria. Purified mitochondria were then exposed ex vivo to 4-HNE for 15 min at 37 °C, which we showed to cause a concentration-related inhibition of mitochondrial respiration together with covalent binding of 4-HNE to mitochondrial proteins. We chose a 30 μM concentration of 4-HNE, which produced an approximately 50% inhibition of complex I- or complex II-driven respiration, to assess whether prior in vivo Nrf2–ARE-activating compounds would increase the resistance of the isolated cortical mitochondria to 4-HNE's mitotoxic effects. Administration of either compound significantly increased (p<0.05) expression of heme oxygenase-1 mRNA in cortical tissue 48 h postadministration, verifying that both compounds were capable of inducing the Nrf2–ARE pathway. Moreover, the prior in vivo administration of SFP and CA significantly (p<0.05) attenuated 4-HNE-induced inhibition of mitochondrial respiration for complex I, but only carnosic acid acted to protect complex II. Furthermore, both CA and SFP significantly (p<0.05) reduced the amount of 4-HNE bound to mitochondrial proteins as determined by Western blot. These results demonstrate the capability of in vivo Nrf2–ARE induction to protect from 4-HNE toxicity to cortical mitochondria ex vivo. Ongoing studies will determine the therapeutic efficacy of Nrf2–ARE activators to attenuate traumatic brain injury-induced pathophysiology.