How root respiration acclimates to global warming remains unclear, especially in subtropical forests that play a key role in the global carbon budget. In a large-scale in situ soil warming experiment, the occurrence of, and mechanisms controlling over, the acclimation of fine-root respiration of Cunninghamia lanceolata during the fourth year of warming were investigated. Specific respiration rates (at reference temperature of 20°C; SRR20 ) were measured with exogenous glucose addition, uncoupler addition, or no addition, and root morphological and chemical traits were also measured. Warming decreased SRR20 by 18.4% only during summer, indicating partial thermal acclimation of fine-root respiration under warming. Warming did not change fine-root N concentration, showing no possible enzyme limitation on respiration. Warming decreased root soluble sugar/starch ratio in summer, and glucose addition increased respiration only under warming, indicating a warming-induced substrate limitation on respiration. Uncoupler addition also stimulated respiration only under warming, showing a warming-induced adenylate limitation on respiration. These findings suggest that thermal acclimation of root respiration in subtropical forests, which is at least partially constrained by substrate and adenylate use, is conducive to reducing ecosystem carbon emissions and mitigating the positive feedback between atmospheric CO2 and climate warming.