Soil microbial autotrophs play an important role in CO₂ fixation in terrestrial ecosystems, particularly in vegetation-constrained ecosystems with environmental stresses, such as the Tibetan Plateau, which is characterized by low temperature, drought, and high UV. However, soil microbial autotrophic communities and their driving factors remain less appreciated in these terrestrial ecosystems. To understand carbon sequestration by soil microbial autotrophs and the carbon cycle in alpine meadow soils on the Tibetan Plateau, autotrophic microbial abundance, community structure, diversity, and their driving environmental factors were explored along an elevation gradient from 4400m to 5200m on the Tibetan Plateau. Additionally, the seasonal change in soil microbial autotrophs was explored at each elevation. The autotrophic microbial communities were characterized by quantitative PCR, T-RFLP, and cloning/sequencing methods targeting four types of cbbL gene, encoding the large subunit for the CO₂-fixing protein ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO), Form IA/B, IC, and ID. High cbbL gene abundance was observed, which significantly increased with increasing elevations, with minimum seasonal change. Among the four types of microbial autotrophs, Form IC gene abundance was the highest at the level of 10⁸ copies/g soil, followed by Form IA/B and Form ID. Form IC cbbL gene abundance was positively correlated with elevation, soil water content (SWC), and NH₄⁺ content, and negatively correlated with soil temperature and pH (P < 0.01). Form IC Shannon diversity and richness significantly increased with increasing elevation, peaking at 4800m, but showed little effect of season. Form IC community structure exhibited a gradual shift along the elevation gradient, and was mainly driven by SWC, soil pH, and elevation. Form IC cbbL genes were identified to be Actinobacteria and Proteobacteria (including α-proteobacteria and β-proteobacteria) using clone library and sequencing, and the dominant phyla were α-proteobacteria in alpine meadow soils, including Bradyrhizobium, Rhizobium, and Mesorhizobium. Our results demonstrated high abundance of microbial autotrophs and high CO₂ fixation potential in alpine grassland soils, and provide a novel model to identify dominant drivers of soil microbial communities and their ecological functions. These findings have important implications for understanding the functional role of microorganisms in the soil carbon cycle and could be helpful to more accurately assess carbon storage in alpine grassland soils on the Tibetan Plateau.