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While many studies have examined the role of biochar in carbon (C) accrual in short-term scale, few have explored the decadal scale influences of biochar on non-biochar C, e.g., native soil organic C (SOC) and added substrate. To address this knowledge gap, soils were collected from decade-old biochar field trials located in the United Kingdom (Cambisol) and China (Fluvisol), with each site having had three application rates (25–30, 50–60 and 75–100 Mg ha−1) of biochar plus an unamended Control, applied once in 2009. We assessed physicochemical and microbial properties associated with sucrose (representing the rhizodeposits) mineralization and the priming effect (PE) on native SOC. Here, we showed both soils amended with biochar at the middle application rate (50 Mg ha−1 biochar in Cambisol and 60 Mg ha−1 biochar in Fluvisol) resulted in greater substrate mineralization. The enhanced accessibility and availability of sucrose to microorganisms, particularly fast-growing bacterial genera like Arenimonas, Spingomonas, and Paenibacillus (r-strategists belonging to the Proteobacteria and Firmicutes phyla, respectively), can be attributed to the improved physicochemical properties of the soil, including pH, porosity, and pore connectivity, as revealed by synchrotron-based micro-CT. Random forest analysis also confirmed the contribution of the microbial diversity and physical properties such as porosity on sucrose mineralization. Biochar at the middle application rate, however, resulted in the lowest PE (0.3 and 0.4 mg of CO2-C g soil−1 in Cambisol and Fluvisol, respectively) after 53 days of incubation. This result might be associated with the fact that the biochar promoted large aggregates formation, which enclosed native SOC in soil macro-aggregates (2–0.25 mm). Our study revealed a diverging pattern between substrate mineralization and SOC priming linked to the biochar application rate. This suggests distinct mechanisms, biophysical and physicochemical, driving the mineralization of non-biochar carbon in a field where biochar was applied a decade before. Highlights: The application of biochar at a moderate rate (50–60 Mg ha−1) resulted in increased labile C mineralization and decreased priming of native SOC. Biochar amendments led to the reconfiguration of physicochemical properties, including pH, porosity, and the formation of aggregates. Modified microbial community structure towards more r-strategists caused greater sucrose mineralization. Biochar incorporation rate governed the C dynamics via (biophysical vs physicochemical mechanisms) in long-term biochar amended fields. [ABSTRACT FROM AUTHOR]