Mammalian neuroglobin (Ngb) is involved in neuroprotection under oxidative stress conditions such as ischemia and reperfusion. However, the neuroprotective mechanism remains unclear. We previously demonstrated that human ferric Ngb binds to the alpha-subunits of heterotrimeric G proteins (Got(i/o)) and acts as a guanine nucleotide dissociation inhibitor (GDI) for G alpha(i/o).. In the present study, we used a protein delivery reagent, Chariot, to investigate whether the GDI activity of human Ngb plays an important role in its neuroprotective activity under oxidative stress conditions. We showed that human Ngb mutants, which retained GDI activities, rescued pheochromocytoma PC12 cell death caused by hypoxia/rcoxygenation as did human wild-type Ngb. In contrast, zebrafish Ngb and human Ngb mutants, which did not function as GDI proteins, did not rescue cell death. These results clearly show that the GDI activity of human Ngb is tightly correlated with its neuroprotective activity. (c) 2008 Elsevier Inc. All rights reserved.