Accurately assessing the efficiency of microbial carbon (C) conversion and accumulation associated with soil “microbial C pump” (MCP) is essential for understanding microbial-mediated soil C sequestration. Conventional assessments based on microbial C use efficiency (CUE) hinge on living biomass only and do not include microbial necromass, which may not depict microbial C accumulation. Here we propose a simple and rapid approach based on 13C-glucose amendment experiment to assess microbial C accumulation efficiency (CAE) in a relatively short term. We first validated the approach by showing negligible sorption of glucose to soils with a wide range of edaphic properties. Glucose-derived 13C may hence be considered to represent microbial C (including biomass and residues) after a few days of addition, given the rapid uptake of glucose by microbes. Microbial CAE may thus be assessed as the recovery of glucose-derived 13C in the soil. By further conducting a meta-analysis of literature data involving isotopically labeled glucose amendment experiments, we revealed distinct variation patterns and influencing factors of CAE and CUE across various terrestrial ecosystems. Compared to CUE, which is mainly regulated by factors influencing microbial physiological processes (particularly substrate availability), CAE is jointly regulated by factors that influence microbial growth and residue persistence (e.g., clay content). These findings underscore that CAE is decoupled from CUE. Given the potential divergence in microbial biomass and residue responses to environmental perturbations, CAE provides a more accurate measure of microbial C conversion and accumulation efficiency than CUE, enabling a clearer understanding of MCP dynamics under global changes.