Brown adipose tissue (BAT) has abundant mitochondria with the unique capability of generating heat via uncoupled respiration. Mitochondrial uncoupling protein 1 (UCP1) is activated in BAT during cold stress and dissipates mitochondrial proton motive force generated by the electron transport chain to generate heat. However, other mitochondrial factors required for brown adipocyte respiration and thermogenesis under cold stress are largely unknown. Here, we show LETM1 domain-containing protein 1 (LETMD1) is a BAT-enriched and cold-induced protein required for cold-stimulated respiration and thermogenesis of BAT. Proximity labeling studies reveal that LETMD1 is a mitochondrial matrix protein.Letmd1knockout male mice display aberrant BAT mitochondria and fail to carry out adaptive thermogenesis under cold stress.Letmd1knockout BAT is deficient in oxidative phosphorylation (OXPHOS) complex proteins and has impaired mitochondrial respiration. In addition, BAT-specificLetmd1deficient mice exhibit phenotypes identical to those observed inLetmd1knockout mice. Collectively, we demonstrate that the BAT-enriched mitochondrial matrix protein LETMD1 plays a tissue-autonomous role that is essential for BAT mitochondrial function and thermogenesis.