Cultured neurons tolerate low H2O2 concentrations (≤50 μM) through the activity of constitutive antioxidant response elements (ARE). At H2O2 levels (≥100 μM), neurons increase expression of the gene encoding for inducible hemoxygenase-1 while superoxide dismutase-2 and catalase remain unchanged. Despite this adaptive response, the endogenous antioxidant systems are overwhelmed, leading to decreased viability. Elevating the neuronal cell content of human neuroglobin (Ngb) prior to insult with 100 or 200 μM H2O2 enhanced cell viability and this resulted in a significant decrease in oxidative stress and an increase in the intracellular ATP concentration, whereas in parental cells exposed to the same H2O2-insult, oxidative stress and ATP increased and decreased, respectively. The mechanism for this increase in ATP involves sustained activation of the mito-KATP channel and an increase in phosphoinositide-3 kinase (PI3K)-mediated phosphorylation of Akt. Pharmacological inhibitors directed toward PI3K (wortmannin and LY294002), or the mito-KATP channel (glybenclamide) inhibited the H 2O2-mediated increase in ATP in cells overexpressing human Ngb and consequently cell viability decreased. Neuroglobin's ability to bolster the intracellular pool of ATP in response to added H2O2 is central to the preservation of cytoskeletal integrity and cell viability.