A key pathological feature of Alzheimer's disease (AD) is the accumulation of the neurotoxic amyloid beta (Aβ) peptide within the brains of affected individuals. Previous studies have shown that neuronal cells selected for resistance to Aβ toxicity display a metabolic shift from mitochondrial-dependent oxidative phosphorylation (OXPHOS) to aerobic glycolysis to meet their energy needs. The Src homology/collagen (Shc) adaptor protein p66Shc is a key regulator of mitochondrial function, ROS production and aging. Moreover, increased expression and activation of p66Shc promotes a shift in the cellular metabolic state from aerobic glycolysis to OXPHOS in cancer cells. Here we evaluated the hypothesis that activation of p66Shc in CNS cells promotes both increased OXPHOS and enhanced sensitivity to Aβ toxicity. The effect of altered p66Shc expression on metabolic activity was assessed in rodent HT22 and B12 cell lines of neuronal and glial origin respectively. Overexpression of p66Shc repressed glycolytic enzyme expression and increased both mitochondrial electron transport chain activity and ROS levels in HT22 cells. The opposite effect was observed when endogenous p66Shc expression was knocked down in B12 cells. Moreover, p66Shc activation in both cell lines increased their sensitivity to Aβ toxicity. Our findings indicate that expression and activation of p66Shc renders CNS cells more sensitive to Aβ toxicity by promoting mitochondrial OXPHOS and ROS production while repressing aerobic glycolysis. Thus, p66Shc may represent a potential therapeutically relevant target for the treatment of AD.