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Apoptosis antagonizing transcription factor (AATF) exerts an effect against oxidative stress, DNA damage and cellular apoptosis. However, its role in neuronal ischemia or hypoxia damage has not been elucidated yet. Present study investigated the neuroprotective roles and mechanisms of AATF under ischemia and hypoxia in vivo and in vitro. Focal cerebral ischemia of rat was generated by distal middle cerebral artery occlusion (dMCAO) model, SH-SY5Y cells were used to generate oxygen glucose deprivation (OGD) model in vitro. Western blot and immunofluorescent staining were used to investigate the expression changes of AATF. CCK-8 and LDH were performed to evaluate cellular survival and cytotoxicity. Overexpression and interference lentivirus vectors were performed to regulate the expression of AATF in SH-SY5Y cells. DHE staining that measured by flow cytometry was performed to investigate cellular superoxide anion levels. 8-OHdG expression and AP sites measurement were used to evaluate DNA damage. DNA Ladder and TUNEL staining were employed to evaluate DNA fragmentation. MNNG and DPQ were respectively used to agitate or antagonist caspase-3 independent PCD (programmed cell death) pathway, STS and Z-VAD-fmk were respectively used to agitate or antagonist caspase-3 dependent PCD pathway. Western blot was performed to investigate the expression of Poly(ADP-ribose) polymers (PAR) and apoptosis inducing factor (AIF) in different cellular components, Co-IP (co-immunoprecipitation) was used to test the interaction of AIF, H2AX and CypA (Cyclophilin A). We found that AATF was increased in cortical neurons after brain ischemia (P<0.001). Besides, AATF was upregulated in OGD-treated SH-SY5Y cells in a time-dependent manner (P=0.007). Additionally, overexpressing AATF ameliorated OGD-induced cellular death (P < 0.001) and cytotoxicity (P = 0.001), and AATF interference exacerbated OGD-induced cellular death (P=0.033) and cytotoxicity (P=0.006). We also found that AATF overexpression suppressed cellular DNA fragmentation (P=0.003) but did not ameliorate oxidative stress and DNA damage. Moreover, we discovered that overexpressing AATF suppressed PAR/AIF signaling pathway via binding with AIF.