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Crop residues and their derived biochar are frequently used for their potential to improve grain yield, soil fertility and carbon (C) sequestration. However, the effects of root are often overlooked, and the effects of chemical fertilizer (NPK) combined with root or its biochar on microbial community structure need further study. This study used 13C-labeled maize root, its biochar and soil with different fertilization for 8 years as materials and substrates. A 112-day incubation experiment was conducted to explore the effects of microbial community on the C processing. During incubation, the root-C (54.9%) mineralized significantly more than biochar-C (12.8%), while NPK addition significantly increased the root-C mineralization. Adding biochar alone did not significantly change the microbial community. Compared to the biochar treatment (BC), the root treatment (R) notably increased the contents of total phospholipid fatty acids (PLFAs), 13C-PLFA and the proportion of fungi and Gram-negative bacteria, but reduced the proportion of actinomycetes. The root mineralization was significantly correlated with the relative content of 13C-Gram-positive bacteria and 13C-fungi, while biochar mineralization was significantly correlated with the relative content of 13C-Gram-positive bacteria and 13C-actinomycetes. Notably, NPK addition significantly increased the contribution of biochar-C to PLFA-C pool, while decreasing the contribution of root-C. In summary, due to microbial adaptation to the lack of bioavailable C in biochar-amended soil, biochar can act as a buffer against the significant disturbance caused by NPK to microbial communities and native soil organic carbon (SOC), which contributes to the steady enhancement in soil C storage. Highlights: The addition of biochar alone for 8 consecutive years did not change the composition of the microbial community structure, but the total PLFA content increased significantly compared to the control. NPK addition reduced the proportion of microbial assimilation of root-C, while increasing the proportion of microbial assimilation of biochar-C. The effect of NPK on microbial biomass is short-lived, but the effect on microbial community structure is long-lasting. Biochar has a stronger buffering effect on the drastic changes in microbial communities and native SOC caused by NPK. [ABSTRACT FROM AUTHOR]