<p>Priming effect (PE) of exogenous organic matter on native soil organic carbon (SOC) is a key driver of soil carbon (C) dynamics. However, how biochar regulates PE and microbial mechanisms remains unclear, limiting accurate assessment of soil C sequestration. We conducted a 360-day incubation experiment with ¹³C labeling to investigate PE dynamics and underlying microbial mechanisms induced by two types of 500&#xa0;°C biochar (rice straw (RB500) and poplar twig (PB500)) in two contrasting soils: high-fertility (SH) and low-fertility (DT). Six treatments were included: S-CK, S-RB500, S-PB500, D-CK, D-RB500 and D-PB500. Initial negative PE was observed in all biochar-amended soils. Positive PE occurred mainly at the mid-incubation (~ 60–120 d) in SH soil and at the late incubation (&gt; 180 d) in DT soil. Furthermore, PB500 induced an earlier transition from negative to positive PE than RB500 in both soils. In SH soil, compared with the early and late incubation stages, S-PB500 exhibited the highest C: N imbalance (4.43) and specific activities of N-acetyl-β-D-glucosaminidase and leucine aminopeptidase (0.20 and 4.89 µmol g C^− 1 h^− 1, respectively) at the mid-incubation stage, and these patterns were more consistent with “microbial N mining” In contrast, both D-RB500 and D-PB500 in the DT soil showed the lowest C: N imbalance (0.24–0.45) and the highest microbial biomass C during the late incubation stage, matching the patterns of “microbial stoichiometric decomposition”. This study proposes potential mechanisms for biochar-induced positive PE in contrasting forest soils and offers new insights into biochar use and soil C sequestration.</p>