Abstract: Accumulation and distribution mechanism of nitrogen in soil particlesize during soil fertility development is of great significance for sustainable agricultural management practices. Based on the longterm fertilization experiment, soil particlesize fractionation was separated by supersonic and centrifugation to explore the impact of longterm fertilization on the soil nitrogen concentration and distribution. Results showed that, after twenty years of fertilization, total nitrogen concentration (TN) under all treatments ranged from 1.19 g/kg to 1.98 g/kg in the 0-20 cm layer. Compared to the CK, TN was increased significantly under all treatments. At the 20-40 cm soil layer, TN varied from 0.59 g/kg to 0.84 g/kg for all treatments, which was obviously lower than that of the 0-20 cm surface soil; Nitrogen concentration in the sand, coarse silt, fine silt, and clay particlesize fractions for the treatments combined with manure were increased by 220%, 84%, 46%, and 26% compared to the NPK treatment with the same amount of nitrogen fertilizer in 0-20 cm soil layer. In 20-40 cm soil layer, compared to the NPK, nitrogen concentration in finesilt and clay particles were improved, whereas nitrogen concentration declined in coarsesilt particles under the treatments combined with organic manure; Soil nitrogen mainly associated with clay particles under the 0-20 cm layer. While in the 20-40 cm layer, there was much higher proportion of nitrogen associated with coarsesilt fraction under the CK, NPK and NPKS, clay particle contained the main part of nitrogen under the treatments with organic manure. There was significant positive linear correlation between crop grain yield and soil nitrogen concentration in the 5 m size fractions under top soil. In 0-20 cm layer, application of organic manure can significantly increase soil nitrogen concentration in bulk soil and particlesize fractions. Soil nitrogen was mainly associated with clay particle in red upland soil for all the treatments. In the 20-40 cm soil layer, application of mineral fertilizers with and without straw incorporation has no effects on nitrogen accumulation in clay size faction. Manure application greatly promotes the nitrogen accumulation in clay size fraction, acting as a sustainable soil nitrogen management practice.