<p>Denitrification inhibitors (DIs) effectively mitigate agricultural soil N2O emissions, but their influence on soil carbon dynamics, particularly the priming effect (PE), is poorly understood. This study examined how the biological denitrification inhibitor (BDI) procyanidins modulates PE and microbial mechanisms in acidic tea plantation soil under nitrogen (N) addition. We conducted a 14-day laboratory incubation with eight treatments: Control, procyanidins (BDI) alone, three rates of N fertilizer alone, and their combinations. We measured soil CO₂ emissions to calculate the PE, alongside soil physicochemical properties, bacterial community composition, and predicted functional profiles. Combined N + BDI reduced cumulative CO₂ emissions (75.36-79.82 mg C kg^-1) relative to the control (88.62 mg C kg^-1), despite higher dissolve organic carbon (DOC). It consistently induced negative PE (-13.26 to -8.80 mg C kg^-1), indicating suppressed native soil organic matter (SOM) decomposition. Negative PE initially intensified then weakened, correlating negatively with bacterial community composition and functional potential (especially carbon-cycling genes) but positively with certain physicochemical properties. Notably, N + BDI downregulated chitinase gene <i>chiA</i> and upregulated carbon-fixation gene <i>aclB</i>, with enrichment of oligotrophic, high-carbon use efficiency (CUE) taxa (e.g., <i>Acidobacteria</i>, <i>Chloroflexi</i>). Co-application of N and BDI drives a microbial shift from “nitrogen mining” of native soil organic matter (SOM) to preferential labile carbon use and enhanced fixation. Evidenced by <i>chiA</i> downregulation, <i>aclB</i> upregulation, and high-CUE community restructuring, this elevates microbial CUE, strengthens the microbial carbon pump, and generates robust negative PE. Procyanidins thus offer dual potential for mitigating N₂O and CO₂ while enhancing carbon retention in acidic agroecosystems. </p> Graphical Abstract <p></p>