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Transferring soil organic carbon (SOC) to the SOC pool fractions is an essential process in soil carbon sequestration. How the application of nitrogen (N) fertilizers can change the SOC functional groups and affect the SOC turnover in the microorganism-driven SOC pool are unclear. A 4-year field experiment with four application rates of N fertilizers (0, 120, 240, and 360 kg N ha−1) was conducted in a rice–wheat rotation system. Applying N fertilizers markedly increased the phospholipid fatty acids of bacteria by 25.3–50.2 % and actinomycetes by 11.1–29.5 %, with larger variations at a higher application rate of N fertilizers compared with no N fertilizer. N fertilization decreased the fungi-to-bacteria ratio. The application of N fertilizers increased O-alkyl C proportion by 18.0–30.7 % and labile SOC content (carbohydrates by 28.9–50.0 %, protein by 23.1–43.0 %, and lipids by 41.5–53.7 %) but decreased aromatic C proportion by 16.0–31.3 %. N fertilizers significantly enhanced the SOC stock in the intra-microaggregate organic matter (iPOM) by 11.4–43.2 %. The highest SOC stock in iPOM was observed at 240 kg N ha−1. The structural equation modeling showed that applying N fertilizers improved the labile SOC functional groups and chemical compounds, and promoted the SOC sequestration in more stable fractions by improving soil microbial abundance. • Nitrogen fertilizers increased bacterial PLFAs, but did not affect fungal PLFAs. • Nitrogen fertilizers decreased aromaticity but improved labile soil organic carbon. • Nitrogen fertilizers promoted the transfer of SOC to more stable aggregate fractions. [ABSTRACT FROM AUTHOR]