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CO₂ fixing microorganisms play a crucial role in carbon sequestration in forest soils, but little is known about the vertical zone differences in their CO₂-fixing potential and the influencing factors. Here, in the soils at three altitudes (600 m, 1200 m, 1500 m) and three depths (0-15 cm, 15-30 cm, 30-45 cm) of Jiugong Mountain, we quantified CO₂ fixation potential through a ¹³C-CO₂ labelling experiment and identified the functional microorganisms and metabolic pathways using metagenomics.