Abstract: In agricultural ecosystems, the application of chemical fertilizer nitrogen is an important management measure to ensure high and stable grain yield and maintain soil nitrogen stability. Soil microorganisms are both drivers of soil nitrogen mineralization and contributors to soil nitrogen retention, playing an active role in nitrogen storage and supply, directly affecting crop nitrogen absorption and utilization. This study is based on the 14-year long-term different nitrogen application level positioning experiment of wheat-corn rotation farmland in Luancheng Agricultural Ecosystem Experimental Station, Chinese Academy of Sciences. It selected three typical treatments of corn season nitrogen application amounts of 150 kg(N)∙hm⁻² (N150), 200 kg(N)∙hm⁻² (N200) and 300 kg(N)∙hm⁻² (N300). By applying ¹⁵N labeled nitrogen fertilizer in micro-regions, the corn yield, total nitrogen absorption of the above-ground part, and fertilizer nitrogen absorption were measured at harvest, and the contents of total nitrogen (TN), microbial residue nitrogen MRN, including fungal residue nitrogen (FRN) and bacterial residue nitrogen (BRN), fixed ammonium (FN), mineral nitrogen (NH₄⁺-N+NO₃⁻-N, MN) and other organic nitrogen (ON) in the 0~20 cm soil layer and the interception of ¹⁵N by different nitrogen pools were analyzed. Through multiple regression analysis and path analysis, the correlation between various forms of nitrogen pools and corn nitrogen absorption was established. The distribution of soil "old nitrogen" and fertilizer "new nitrogen" in soil nitrogen pools and their effects on crop absorption and utilization were studied, providing theoretical support for the efficient use of nitrogen fertilizer and soil fertility cultivation in the study area. The results showed that corn yield, nitrogen absorption and soil TN content were all highest at N200, which is conducive to high crop yield and soil nitrogen pool cultivation. The absorption and residual amount of fertilizer nitrogen at N300 were higher than those at N200, indicating that the soil nitrogen "stimulation effect" at N300 was stronger than that at N200, which would induce more mineralization and loss of soil "old nitrogen", and its soil TN pool stability was poor and the renewal degree was larger. Among the total nitrogen pools, MRN was dominant, N200 was significantly higher than other treatments, contributing more than 50% to TN, and FRN dominated the accumulation of MRN. The soil FRN:BRN ratio of N200 was significantly higher than that of N150 and N300, indicating that appropriate nitrogen application can significantly increase the contribution of fungi to nitrogen accumulation and improve the stability of soil nitrogen pool; insufficient nitrogen application (N150) or excessive nitrogen application (N300) increased the contribution of bacteria to nitrogen accumulation, which is not conducive to the stability of soil nitrogen pool. The MN and FN content of N300 was significantly higher than other treatments, indicating that excessive fertilization mainly increases the active nitrogen pool. Therefore, appropriate nitrogen application can optimize the distribution of soil nitrogen pool, promote more nitrogen into the microbial residue nitrogen pool, significantly improve the nitrogen retention function of soil microbial pathway, promote the benign operation of soil nitrogen storage and supply, ensure the formation of corn nitrogen absorption and yield, and provide scientific basis for guiding soil fertility cultivation and nitrogen fertilizer reduction in the North China Plain.ain.