The study investigates the role of adenosine monophosphate-activated protein kinase (AMPK) in mediating mitochondrial fission in response to energy stress. AMPK is genetically required for rapid mitochondrial fragmentation after treatment with electron transport chain (ETC) inhibitors, and its direct pharmacological activation also promotes mitochondrial fragmentation. The mitochondrial fission factor (MFF), a receptor for DRP1, is identified as a key effector of AMPK-mediated mitochondrial fission. MFF contains two predicted AMPK phosphorylation sites, Ser155 and Ser172, which are essential for its function. Phosphorylation of these sites by AMPK is necessary for the recruitment of DRP1 to mitochondria and subsequent mitochondrial fission. The study demonstrates that AMPK-dependent phosphorylation of MFF is crucial for the rapid induction of mitochondrial fission in response to energy stress, potentially facilitating the initiation of mitophagy and the biogenesis of new mitochondria.The study investigates the role of adenosine monophosphate-activated protein kinase (AMPK) in mediating mitochondrial fission in response to energy stress. AMPK is genetically required for rapid mitochondrial fragmentation after treatment with electron transport chain (ETC) inhibitors, and its direct pharmacological activation also promotes mitochondrial fragmentation. The mitochondrial fission factor (MFF), a receptor for DRP1, is identified as a key effector of AMPK-mediated mitochondrial fission. MFF contains two predicted AMPK phosphorylation sites, Ser155 and Ser172, which are essential for its function. Phosphorylation of these sites by AMPK is necessary for the recruitment of DRP1 to mitochondria and subsequent mitochondrial fission. The study demonstrates that AMPK-dependent phosphorylation of MFF is crucial for the rapid induction of mitochondrial fission in response to energy stress, potentially facilitating the initiation of mitophagy and the biogenesis of new mitochondria.