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Soil net nitrogen mineralization rate (N-min), which is critical for soil nitrogen availability and plant growth, is thought to be primarily controlled by climate and soil physical and/or chemical properties. However, the role of microbes on regulating soil N-min has not been evaluated on the global scale. By compiling 1565 observational data points of potential net N-min from 198 published studies across terrestrial ecosystems, we found that N-min significantly increased with soil microbial biomass, total nitrogen, and mean annual precipitation, but decreased with soil pH. The variation of N-min was ascribed predominantly to soil microbial biomass on global and biome scales. Mean annual precipitation, soil pH, and total soil nitrogen significantly influenced N-min through soil microbes. The structural equation models (SEM) showed that soil substrates were the main factors controlling N-min when microbial biomass was excluded. Microbe became the primary driver when it was included in SEM analysis. SEM with soil microbial biomass improved the N-min prediction by 19% in comparison with that devoid of soil microbial biomass. The changes in N-min contributed the most to global soil NH4+-N variations in contrast to climate and soil properties. This study reveals the complex interactions of climate, soil properties, and microbes on N-min and highlights the importance of soil microbial biomass in determining N-min and nitrogen availability across the globe. The findings necessitate accurate representation of microbes in Earth system models to better predict nitrogen cycle under global change.