The study investigates the mechanism by which mitochondrial protein lactylation limits oxidative phosphorylation (OXPHOS) under hypoxic conditions. Mitochondrial alanyl-tRNA synthetase (AARS2) is identified as a protein lysine lactyltransferase that is upregulated in response to hypoxia. AARS2 lactylates PDHA1 at lysine 336 and CPT2 at lysine 457/8, inactivating these enzymes and inhibiting OXPHOS. This lactylation can be reversed by SIRT3, which deactylates the lactylated lysine residues. During exercise, lactate oxidation-induced intracellular hypoxia leads to increased lactylation, which constrains high-intensity endurance running performance. The study reveals that mitochondrial protein lactylation integrates intracellular hypoxia and lactate signals to regulate OXPHOS, providing a feedback mechanism to balance OXPHOS activity and intracellular oxygen availability.The study investigates the mechanism by which mitochondrial protein lactylation limits oxidative phosphorylation (OXPHOS) under hypoxic conditions. Mitochondrial alanyl-tRNA synthetase (AARS2) is identified as a protein lysine lactyltransferase that is upregulated in response to hypoxia. AARS2 lactylates PDHA1 at lysine 336 and CPT2 at lysine 457/8, inactivating these enzymes and inhibiting OXPHOS. This lactylation can be reversed by SIRT3, which deactylates the lactylated lysine residues. During exercise, lactate oxidation-induced intracellular hypoxia leads to increased lactylation, which constrains high-intensity endurance running performance. The study reveals that mitochondrial protein lactylation integrates intracellular hypoxia and lactate signals to regulate OXPHOS, providing a feedback mechanism to balance OXPHOS activity and intracellular oxygen availability.