Calcareous alkaline soils are widely distributed in arid and semi-arid regions, and generally exhibit low soil organic carbon (SOC) sequestration capacity. Although calcium carbonate (CaCO3) may have an impact on the organic carbon stabilization in these soils, the specific mechanisms remain unclear. Through a 180-day pot experiment incorporating five graded levels of CaCO3, this study investigated its impact on SOC sequestration combined with detailed mineralogical and microbiological analyses. Results indicated that high-calcareous soil significantly decreased SOC from 14.07 to 4.75 g kg- 1, particulate organic carbon from 4.34 to 0.89 g kg- 1, and mineral-associated organic carbon from 8.27 to 2.62 g kg- 1. Microbial necromass carbon was greatly reduced with high-calcareous soil. The structure and functional metabolism of soil microbial communities were altered. Compared to low-calcareous soils, high-calcareous soils reduced microbial biomass, diversity, and fungal relative abundance; Bacteroidota and Ascomycota replaced Acidobacteriota and Mucoromycota as dominant taxa. Mineralogical analyses indicated that CaCO3 coating on soil mineral surfaces reduced the specific surface area by 0.64-4.86 fold, attenuating the organo-mineral associative capacity. In summary, SOC sequestration in calcareous soils may be suppressed through a dual mechanism involving the suppression of mineral surface reactivity and the modulation of microbial community structure and function. This study provides novel mechanistic insights into carbon sequestration constraints in calcareous environments.