Autophagy is a physiological process by which the cell eliminates damaged organelles, toxic agents, and long-lived proteins by degradation through lysosomal system. Mitophagy, the specific autophagic elimination of mitochondria, regulates mitochondrial number to match metabolic demand and is a core machinery of quality control to remove damaged mitochondria. A neuroprotective role of physiological autophagy/mitophagy has been discovered. However, recent studies suggested that highly accelerated autophagy/mitophagy might contribute to neuronal death in various pathological situations including cerebral ischemia. In this study, we aimed to investigate the activation of excessive autophagy, particularly, the more specific mitophagy, in neuronal tissues and its contribution to ischemia/hypoxia (I/H)-induced delayed neuronal death. I/H injury was induced by oxygen and glucose deprivation (OGD) followed by reperfusion (RP) on primary cortical neurons in vitro. Cerebral ischemia was induced by unilateral common carotid artery occlusion and hypoxia in neonatal mice in vivo. In order to determine the extent to which autophagy contributes to neuronal death in cerebral ischemia, we performed multiple methods and found that in both primary cortical neurons and SH-SY5Y cells exposed to OGD for 6 h and RP for 24, 48, and 72 h, respectively, an increase of autophagy was observed as determined by the increased ratio of LC3-II to LC3-I and Beclin 1 expression. Using Fluoro-Jade C and monodansylcadaverine double-staining, and electron microscopy we found the increment in autophagy after OGD/RP was accompanied by increased autophagic cell death, and this increased cell death was inhibited by the specific autophagy inhibitor, 3-methyladenine. The presence of large autolysosomes and numerous autophagosomes in cortical neurons were confirmed by electron microscopy. Autophagy activities were increased dramatically in the ischemic brains 3-7 days postinjury from a rat model of neonatal cerebral I/H as shown by increased punctate LC3 staining and Beclin-1 expression. We thus obtained the conclusion that excessive activation of autophagy contributes to neuronal death in cerebral ischemia. BNIP3 (Bcl-2/adenovirus E19 kD interacting protein 3), a member of a unique subfamily of death-inducing mitochondrial proteins, is highly associated with mitochondrial dysfunction and delayed neuronal death in stroke. It is known that BNIP3-induced neuronal death is caspase-independent and characterized by early mitochondrial damage. Recent evidence suggested that the BNIP3 family of proteins might be important regulators of mitophagy. Here, using both stroke models, we found that homodimer (60 kD) of BNIP3/NIX (BNIP3L) were highly expressed in a ‘delayed’ manner. Particularly, significant mitophagic activation was confirmed by electron microscopy. In contrast, both neonatal mitophagy and apoptosis were significantly inhibited in the BNIP3 knockout (KO) mice after I/H, which was also accompanied by a significantly increased autophagic response. In addition, the infarct volume in the BNIP3 KO mice was significantly reduced as compared to wild-type (WT) mice after 7 or 28 days recovery, showing a prominent neuroprotection of BNIP3 gene silencing. A protein-to-protein interaction of mitochondria-localized BNIP3 (60 kD) with the autophagosome marker, LC3, was confirmed by co-ip, immunocytochemistry and further quantified by ELISA, indicating BNIP3 was an effective LC3-binding target on damaged mitochondria. These data demonstrated a novel role of BNIP3 in regulating neuronal mitophagy and cell death during ischemic stroke.