Abstract: In farmland ecosystems, the application of chemical nitrogen (N) fertilizers is a crucial management strategy to ensure stable and high grain yields while maintaining the stability of soil N pools. Soil microorganisms play a pivotal role in both N mineralization and immobilization, thereby actively contributing to N retention and supply, which directly affects N uptake and utilization by crops. This study was conducted in the wheat-maize rotation field of the Luancheng Agro-Ecosystem Experimental Station of the Chinese Academy of Sciences. This study relied on a 14-year long-term positioning experiment with different N application rates. Three typical treatments were selected for N application rates during the maize season: 150 kg(N)∙hm⁻² (N150), 200 kg(N)∙hm⁻² (N200), and 300 kg(N)∙hm⁻² (N300). Micro-plots were used to apply ¹⁵N-labeled N fertilizer to investigate the distribution of “old N” from soil and “new N” from fertilizer within soil N pools and their impact on crop N uptake and utilization. At harvest, the maize yield, total N uptake by the aboveground parts, and N uptake from fertilizers were measured. The contents of total N (TN), microbial residue N (MRN), fixed ammonium (FN), mineral N (NH₄⁺-N + NO₃⁻-N, MN), and other organic N (ON) in the 0−20 cm soil layer were analyzed, along with the interception of ¹⁵N by different N pools. Multivariate regression and path analyses were used to establish the relationships between various N pool forms and maize N uptake. The results indicated that maize yield, N uptake, and soil TN content were the highest under the N200 treatment, favoring high crop yields and soil N pool cultivation. Fertilizer N uptake and residue were higher under the N300 treatment than under the N200 treatment, suggesting a stronger “priming effect” under the N300 treatment, which induces greater mineralization and loss of soil “old N”. The stability of soil TN pool was poor under the N300 treatment, with a higher degree of turnover. Among the TN pools, MRN was dominant, with the N200 treatment being significantly higher than N150 treatment, contributing more than 50% of the TN. Fungal residue N (FRN) was the main contributor to MRN accumulation. The ratio of FRN to bacterial residual N (BRN) was higher in the N200 treatment than in the N150 and N300 treatments, indicating that optimal N application could significantly enhance the contribution of fungi to N accumulation, thereby improving the stability of the soil N pool. Insufficient N application (N150) or excessive N application (N300) increased the contribution of bacteria to N accumulation, which was not conducive to stabilizing the soil N pool. The MN and FN contents were significantly higher in the N300 treatment than in other treatments, suggesting that excessive fertilization predominantly enhanced the active N pool. In conclusion, optimal N application can optimize the soil N pool distribution, promote the sequestration of N into microbial residue pools, significantly enhance the N immobilization capacity of soil microorganisms, and foster the benign operation of soil N retention and supply. This ensures maize N uptake and yield. The study provides a scientific basis for guiding farmland fertility improvement and N fertilizer reduction in the North China Plain.