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• We describe microbial biomass role in C sequestration in fertilized paddy soil. • Organic inputs increase microbial growth and necromass accumulation. • Organic inputs do not alter microbial CUE in bulk soil, but reduce CUE in rhizosphere soil. • Microbial CUE is lower in rhizosphere soil than in bulk soil. Microbial anabolism relative to catabolism, reflected by the C use efficiency (CUE), determines the fate of C transformation in soil. Understanding how the microbial CUE and microbial necromass respond to fertilization is crucial for the evaluation of the C sequestration potential in intensively managed paddy soils. We examined the microbial CUE, microbial biomass turnover, and necromass accumulation in rice rhizosphere and bulk soils subjected to long-term (31 years) fertilizations: no fertilizers (control), mineral fertilizers alone (NPK), mineral fertilizers plus rice straw incorporation (NPK-Straw), and mineral fertilizers combined with a low or a high amount of organic manure (NPK-lowM or NPK-highM). The microbial CUE was determined by 18O incorporation into DNA. Microbial necromass accumulation was quantified by the biomarker analysis of amino sugars. Rice straw and manure incorporation reduced the microbial CUE in the rhizosphere soil, whereas the CUE remained constant in the bulk soil. CUE was lower in the rhizosphere soil than in the bulk soil due to nutrients uptake and root exudate release by rice plants, leading to a higher C/nutrient ratio in the rhizosphere. Organic inputs strengthened these rhizosphere processes and could thus weaken the relative potential of C sequestration. The microbial CUE decreased with the increase of the available C/N ratio in the rhizosphere but not in the bulk soil. The microbial CUE mainly depended on the respiration in the bulk soil and on the microbial growth in the rhizosphere soil, indicating the divergent microbial utilization of organic substrates between rhizosphere and bulk soils. In both rhizosphere and bulk soils, organic inputs promoted the microbial biomass growth rate and further increased the amount of microbial necromass by 27–52 % compared with NPK alone, which was highly correlated with the soil organic C pools. Despite enhancing rhizosphere respiration, our findings highlight that rice straw and manure applications increase C sequestration in paddy soils by enhancing the net flux of microbial biomass formation, and consequently promoting necromass accumulation. [ABSTRACT FROM AUTHOR]