This study aimed to explore the effects of long-term nitrogen fertilizer application on the mineral composition and microbial necromass carbon in the soil profile and its mechanism of action on soil organic carbon sequestration. 【Method】This study was based on a 38-year long-term fixed-site fertilization experiment on brown soil. The treatments of no fertilization (CK) and long-term nitrogen fertilizer application (N) were selected as the research objects. Techniques such as soil physical fractionation, biomarker analysis, and X-ray diffraction (XRD) were comprehensively employed to systematically analyze the variation characteristics of mineral composition. The analysis also extended to evaluating microbial necromass carbon, different carbon pools in the soil profile (0-100 cm), and revealing the accumulation characteristics and driving mechanisms of the soil organic carbon pool under long-term nitrogen fertilizer application. 【Result】 The contents of soil organic carbon, particulate organic carbon, and mineral-associated organic carbon significantly decreased with increasing soil depth, while dissolved organic carbon first increased and then decreased, peaking in the 20-40 cm layer in both CK and N treatments. Nitrogen fertilizer application did not change the mineral types in the soil profile, but had a significant impact on the profile distribution characteristics of minerals. Long-term nitrogen fertilization promoted the accumulation of microbial necromass carbon in the soil profile and its contribution to soil organic carbon, but decreased the fungal-to-bacterial necromass carbon ratio in the 0-60 cm layer while increasing it in the 60-100 cm layer. Also, long-term nitrogen fertilization significantly increased the contribution of particulate organic carbon to soil organic carbon in the 20-60 cm soil layer, while significantly reducing the contribution of mineral-associated organic carbon to soil organic carbon in the 20-40 cm soil layer. 【Conclusion】Although long-term nitrogen fertilizer application can enhance the function of the “microbial carbon pump”, the reduction of montmorillonite, the increase of kaolinite, and the increased proportion of fungal residues in the deep soil collectively resulted in the failure of microbial necromass carbon to synchronously transform into mineral-associated organic carbon accumulation. This indicates an “asynchrony” between mineral-associated organic carbon and mineral protection in the deep soil.