The molecular mechanisms of neuronal cell death following circulatory arrest are still not fully understood. In the current study we investigated the role of apoptosis-inducing factor (AIF), a major caspase-independent mitochondrial cell death protein, for neuronal cell death following global cerebral ischemia (GCI). C57/Bl6 or low AIF expressing Harlequin mutant mice (AIF(low)) and their wild-type littermates were subjected to 10 min of GCI. DNA damage, nuclear pathology, and localization of AIF were investigated 6, 24, and 72 h after GCI by TUNEL and DAPI staining, and immunohistochemistry, respectively. Cell death of hippocampal CA1 neurons following GCI was associated with nuclear translocation of AIF, nuclear pyknosis, and DNA fragmentation, i.e. ∼80% of all TUNEL-positive neurons had nuclear AIF staining. In AIF(low) mice neuronal cell loss was reduced by 60% (p<0.02). The current experiments suggest that AIF-mediated signaling represents a novel mechanism of neuronal cell death following GCI.