AMP-activated protein kinase (AMPK) mediates mitochondrial fission in response to energy stress. This study shows that AMPK is genetically required for rapid mitochondrial fragmentation after treatment with electron transport chain (ETC) inhibitors. Direct activation of AMPK also promotes mitochondrial fragmentation even in the absence of mitochondrial stress. A screen for AMPK substrates identified mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for DRP1, a cytoplasmic GTPase that catalyzes mitochondrial fission. Nonphosphorylatable and phosphomimetic alleles of the AMPK sites in MFF revealed that it is a key effector of AMPK-mediated mitochondrial fission. Metabolic stress that damages mitochondria triggers mitochondrial fragmentation, leading to degradation of defective mitochondria (mitophagy) or apoptosis in cases of severe damage. This response enables the consolidation of the still-intact functional elements of mitochondria, while allowing for physical segregation of dysfunctional mitochondrial components into depolarized daughter organelles that are targeted for mitophagy. Similarly, proper mitochondrial fission facilitates timely apoptosis. Mitochondrial fragmentation is also associated with mitochondrial dysfunction, such as in diseases associated with mitochondrial DNA (mtDNA) mutations. Conversely, mitochondrial fusion is thought to promote oxidative phosphorylation, to spare mitochondria from mitophagy, and to allow biodistribution of fatty acids for fuel utilization under nutrient-limited conditions to maintain metabolite pools and efficient ATP production. AMPK is a central metabolic sensor activated by a wide variety of mitochondrial insults. Under conditions when intracellular ATP concentrations decrease, increased intracellular AMP directly binds to the γ regulatory subunit of AMPK, facilitating AMPK activation by the upstream protein kinase LKB1. Upon activation, AMPK restores intracellular energy levels through direct phosphorylation of multiple downstream substrates that inhibit ATP-consuming biosynthetic pathways and stimulate catabolic ATP-regenerating processes. Because mitochondria supply the majority of cellular ATP, the maintenance of mitochondrial function is critical to maintaining overall energetic homeostasis. However, the question of whether AMPK plays direct roles in different aspects of mitochondrial biology has not been well examined. AMPK has previously been tied to mitochondrial integrity through its direct phosphorylation and activation of the highly conserved autophagy kinase ULK1, which promotes mitophagy. In comparison, although mitochondrial fission and fusion rates are known to respond to changes in cellular metabolism, the molecular details of how changes in cellular bioenergetics and nutrients couple to the fission and fusion machinery remain poorly understood. The study shows that AMPK is required for mitochondrial fragmentation in response to energy stress. AMPK activation leads to mitochondrial fragmentation, and this effect is restored when AMPK is reconstituted in AMPK double knockout cells. The study also shows that MFF is a conserved substrate of AMPK, and that phosphorylation of MFF is required for recruitment of DRP1 to mitochondria after AMPAMP-activated protein kinase (AMPK) mediates mitochondrial fission in response to energy stress. This study shows that AMPK is genetically required for rapid mitochondrial fragmentation after treatment with electron transport chain (ETC) inhibitors. Direct activation of AMPK also promotes mitochondrial fragmentation even in the absence of mitochondrial stress. A screen for AMPK substrates identified mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for DRP1, a cytoplasmic GTPase that catalyzes mitochondrial fission. Nonphosphorylatable and phosphomimetic alleles of the AMPK sites in MFF revealed that it is a key effector of AMPK-mediated mitochondrial fission. Metabolic stress that damages mitochondria triggers mitochondrial fragmentation, leading to degradation of defective mitochondria (mitophagy) or apoptosis in cases of severe damage. This response enables the consolidation of the still-intact functional elements of mitochondria, while allowing for physical segregation of dysfunctional mitochondrial components into depolarized daughter organelles that are targeted for mitophagy. Similarly, proper mitochondrial fission facilitates timely apoptosis. Mitochondrial fragmentation is also associated with mitochondrial dysfunction, such as in diseases associated with mitochondrial DNA (mtDNA) mutations. Conversely, mitochondrial fusion is thought to promote oxidative phosphorylation, to spare mitochondria from mitophagy, and to allow biodistribution of fatty acids for fuel utilization under nutrient-limited conditions to maintain metabolite pools and efficient ATP production. AMPK is a central metabolic sensor activated by a wide variety of mitochondrial insults. Under conditions when intracellular ATP concentrations decrease, increased intracellular AMP directly binds to the γ regulatory subunit of AMPK, facilitating AMPK activation by the upstream protein kinase LKB1. Upon activation, AMPK restores intracellular energy levels through direct phosphorylation of multiple downstream substrates that inhibit ATP-consuming biosynthetic pathways and stimulate catabolic ATP-regenerating processes. Because mitochondria supply the majority of cellular ATP, the maintenance of mitochondrial function is critical to maintaining overall energetic homeostasis. However, the question of whether AMPK plays direct roles in different aspects of mitochondrial biology has not been well examined. AMPK has previously been tied to mitochondrial integrity through its direct phosphorylation and activation of the highly conserved autophagy kinase ULK1, which promotes mitophagy. In comparison, although mitochondrial fission and fusion rates are known to respond to changes in cellular metabolism, the molecular details of how changes in cellular bioenergetics and nutrients couple to the fission and fusion machinery remain poorly understood. The study shows that AMPK is required for mitochondrial fragmentation in response to energy stress. AMPK activation leads to mitochondrial fragmentation, and this effect is restored when AMPK is reconstituted in AMPK double knockout cells. The study also shows that MFF is a conserved substrate of AMPK, and that phosphorylation of MFF is required for recruitment of DRP1 to mitochondria after AMP