Although CO2-fixing microorganisms contribute to the soil organic carbon (SOC) pool via organic metabolites, their roles in driving microbial biomass synthesis, necromass formation, and SOC accumulation across distinct vegetation regions remain unclear. This study aims to clarify the regulatory mechanism of vegetation types on microbial CO2 fixation and SOC accumulation. Based on 13C-CO2 labeling tracing, biomarker analysis, and metagenomic sequencing, we investigated microbial CO2 fixation potential and the allocation of assimilated carbon to microbial biomass carbon (MBC) and microbial necromass carbon (MNC) in grassland, shrub, and forest soils on the Loess Plateau. After 61 days of 13CO2 labeling, SOC content in grassland soils significantly increased, while remaining stable in shrub and forest soils. 13C tracing results revealed that grassland soils exhibited higher 13C enrichment in SOC (delta 13C = 194%o) and a higher contribution of microbial CO2 fixation to SOC (0.21%) compared to the other two soil types. Additionally, the total MBC content significantly increased in all soils after labeling; specifically, shrub soils had higher 13C enrichment in MBC, 13C-CMBC content, and contribution ratio to total MBC than grassland and forest soils. In contrast, bacterial necromass carbon (BNC) contents increased substantially across all sites, while fungal necromass carbon contents decreased. Grassland soils showed prominent 13C enrichment in muramic acid (MurA, delta 13C = 163%o) with a 13C-MurA/MurA ratio of 1.3%, which was superior to those of glucosamine and galactosamine. These results indicated that grasslands, despite low productivity and nutrients, efficiently accumulate fixed CO2 into SOC via bacterial necromass through microbial CO2 fixation. This study confirmed that vegetation type regulated soil properties and microbial communities, which modulate carbon-fixation gene abundance to select specific pathways (reductive citrate/3-hydroxypropionate cycles in nutrient-rich forests vs. Calvin cycle in nutrient-poor grasslands). This differentiation driven distinct 13C allocation patterns and affected SOC accumulation, advancing our understanding of vegetation-regulated terrestrial carbon sequestration mechanisms.