【Objective】Exogenous substrate quality (carbon to nitrogen ratio, C/N) can mediate priming effect (PE). However, the effects and regulation mechanism of priming effect under different C/N ratios of substrate addition is still unclear. 【Method】The effects and regulation mechanism of purple soil which was fertilized by crop straw with synthetic fertilizers were explored through an indoor incubation experiment using glucose and ammonium sulfate. 【Result】Positive priming effect during the incubation period was significantly reduced by 87.4 % and 93.7 % when the material C/N was 10 (CN10) compared to the treatments with a C: N of 50 (CN50) and 100 (CN100). CN100 and CN50 treatments significantly increased soil soluble organic carbon (DOC) and microbial carbon (MBC) content but significantly decreased total soluble nitrogen (TDN) content compared to CN10. After 14 and 43 days of incubation, CN100 treatment significantly elevated the activities of cellobiohydrolase (CBH), β-N-acetylglucosaminoglycosidase (NAG), and leucine aminopeptidase (LAP) compared to CN10. Positive PE in the first two weeks was significantly positively correlated with MBC, CBH, NAG, and LAP, and negatively correlated with TDN, (βG+CBH)/(NAG+LAP) (βG, β-glucosidase), and at the end of the incubation, the positive PE was significantly positively correlated with MBC, βG, CBH, NAG, LAP, and (βG+CBH)/(NAG+LAP) and remained negatively correlated with TDN.【Conclusion】Lower C: N substrate addition significantly reduced the positive PE in purple soils and contributed to efficient soil carbon sequestration; Microorganisms responded to changes in the relative effectiveness of nutrients in the soil environment mainly by adjusting the activities of key enzymes, which in turn regulated the PE. This study can provide a theoretical basis for regional development of fertilizer application programs for efficient carbon sequestration, as well as an in-depth understanding of carbon dynamics in agroecosystems and their microbial-driven mechanisms.