ABSTRACTCarbon use efficiency (CUE) of microbial communities in soil quantifies the proportion of organic carbon (C) taken up by microorganisms that is allocated to growing microbial biomass as well as used for reparation of cell components. This C amount in microbial biomass is subsequently involved in microbial turnover, partly leading to microbial necromass formation, which can be further stabilized in soil. To unravel the underlying regulatory factors and spatial patterns of CUE on a large scale and across biomes (forests, grasslands, croplands), we evaluated 670 individual CUE data obtained by three commonly used approaches: (i) tracing of a substrate C by 13C (or 14C) incorporation into microbial biomass and respired CO2 (hereafter 13C‐substrate), (ii) incorporation of 18O from water into DNA (18O‐water), and (iii) stoichiometric modelling based on the activities of enzymes responsible for C and nitrogen (N) cycles. The global mean of microbial CUE in soil depends on the approach: 0.59 for the 13C‐substrate approach, and 0.34 for the stoichiometric modelling and for the 18O‐water approaches. Across biomes, microbial CUE was highest in grassland soils, followed by cropland and forest soils. A power‐law relationship was identified between microbial CUE and growth rates, indicating that faster C utilization for growth corresponds to reduced C losses for maintenance and associated with mortality. Microbial growth rate increased with the content of soil organic C, total N, total phosphorus, and fungi/bacteria ratio. Our results contribute to understanding the linkage between microbial growth rates and CUE, thereby offering insights into the impacts of climate change and ecosystem disturbances on microbial physiology with consequences for C cycling.