Microbial carbon use efficiency (CUE), a key link between microbial metabolism and ecosystem carbon cycling, is influenced by litter input, nutrient availability, and microbial diversity during vegetation succession. Although microbial carbon use efficiency (CUE) has been widely studied, most existing work has focused on short-term disturbances, laboratory incubations, or surface soils, leaving the depth-specific mechanisms of CUE regulation during long-term vegetation succession insufficiently understood. Here, this study investigated an similar to 170-year vegetation succession chronosequence from grassland to climax forest, analyzing microbial community structure, function, and CUE in topsoil (0-20 cm) and subsoil (20-40 cm) using amplicon sequencing, high-throughput qPCR microarrays, and biogeochemical stoichiometric modeling. The results revealed topsoil CUE remained lower than subsoil CUE consistently during vegetation succession. Topsoil CUE showed an initial increase, followed by a decline and rebound, while subsoil CUE increased initially, then steadily declined. In topsoil, CUE was mainly driven by nutrient availability, indirectly influenced by litter quality, microbial diversity, and microbial products (e.g., biomass, extracellular enzymes). In subsoil, CUE was more directly regulated by nutrients and functional gene abundance (e.g., rbcL, nosZ2, nirS3, nirK2). CUE was shaped by resource inputs (e.g., dissolved organic C and N, litter C:N) and environmental conditions (pH, moisture). Despite pronounced shifts in microbial composition, CUE remained relatively stable in early and late succession. These findings highlight depth-specific microbial processes controlling CUE and provide mechanistic insights into soil carbon stabilization during long-term vegetation succession.