Neuroglobin (Ngb) is a 17 kDa monomeric protein, member of the vertebrate globin family, and so-named for its prevalent expression in neuronal tissue. Currently, Ngb has received great attention as a new neuroprotectant. In vivo and in vitro studies demonstrated that Ngb expression in neurons is up-regulated after neuronal hypoxia, H2O2-induced cell death, and cerebral ischemia. Recently, we demonstrated that 1 nM 17b-estradiol (E2) rapidly induces 300% increase of Ngb levels in neuroblastoma cell line and in primary hippocampal neurons, E2 effect is specific being not induced by testosterone or dihydro-testosterone. These results raise the hypothesis that Ngb is part of E2-induced neuroprotection. Aim of this work was to evaluate the mechanisms underlying E2-induced Ngb levels and the role played by Ngb in E2-induced neuroprotection in neuroblastoma cells. Although E2 rapidly activates p38 and Akt phosphorylation in neuroblastoma cell line, only estrogen receptor b-dependent p38 activation is involved in both rapid (i.e., 1 h) and long term (i.e., 24 h) E2-induced increase of Ngb levels. Similarly, only p38 inhibitor prevents E2-induced Ngb level increase. Intriguingly, E2 induces Ngb translocation from nucleus into mitochondria increasing Ngb-cytochrome c association. Lastly, E2 exerts a protective effect against H2O2-induced neuroblastoma cell death which is completely prevented in Ngb-silenced cells. Overall these data indicate, for the first time, Ngb as an important part of E2 signals involved in hormone-induced protective effects against H2O2-neurotoxicity.