Mitochondrial protein lactylation is induced by intracellular hypoxia to limit oxidative phosphorylation (OXPHOS). This study reveals that mitochondrial alanyl-tRNA synthetase 2 (AARS2), a protein lysine lactyltransferase, is regulated by hypoxia through proline 377 hydroxylation by PHD2. Hypoxia leads to AARS2 accumulation, which lactylates PDHA1 and CPT2, inactivating these enzymes and inhibiting OXPHOS by limiting acetyl-CoA influx. SIRT3 can reverse this lactylation to activate OXPHOS. In mouse muscle cells, lactylation is induced by lactate oxidation during exercise, which can be increased or decreased by adjusting lactylation levels. The findings show that mitochondrial protein lactylation integrates intracellular hypoxia and lactate signals to regulate OXPHOS. AARS2 acts as a substrate of the PHD2-VHL proteasomal machinery, and its inactivation of PDHA1 and CPT2 via lactylation limits OXPHOS. Lactate inactivates PDHA1 and CPT2 dependent on AARS2, and AARS2 lactylates these proteins. SIRT3 reverses lactylation, restoring OXPHOS activity. OXPHOS-induced intracellular hypoxia drives lactylation, which in turn limits OXPHOS and endurance running ability. The study highlights the role of mitochondrial protein lactylation in regulating OXPHOS and endurance exercise.Mitochondrial protein lactylation is induced by intracellular hypoxia to limit oxidative phosphorylation (OXPHOS). This study reveals that mitochondrial alanyl-tRNA synthetase 2 (AARS2), a protein lysine lactyltransferase, is regulated by hypoxia through proline 377 hydroxylation by PHD2. Hypoxia leads to AARS2 accumulation, which lactylates PDHA1 and CPT2, inactivating these enzymes and inhibiting OXPHOS by limiting acetyl-CoA influx. SIRT3 can reverse this lactylation to activate OXPHOS. In mouse muscle cells, lactylation is induced by lactate oxidation during exercise, which can be increased or decreased by adjusting lactylation levels. The findings show that mitochondrial protein lactylation integrates intracellular hypoxia and lactate signals to regulate OXPHOS. AARS2 acts as a substrate of the PHD2-VHL proteasomal machinery, and its inactivation of PDHA1 and CPT2 via lactylation limits OXPHOS. Lactate inactivates PDHA1 and CPT2 dependent on AARS2, and AARS2 lactylates these proteins. SIRT3 reverses lactylation, restoring OXPHOS activity. OXPHOS-induced intracellular hypoxia drives lactylation, which in turn limits OXPHOS and endurance running ability. The study highlights the role of mitochondrial protein lactylation in regulating OXPHOS and endurance exercise.