The purpose of this study was to explore the effect and its mechanism of long-term nitrogen application on the characteristics of endophytic fungi community in crop transport roots and absorbing roots. Based on the field long-term location fertilization experiment (1995-2019), through the high-throughput sequencing technology, the long-term 5 nitrogen fertilizer treatments (CK, N1, N2, N1P, N2P) were studied. Among them, the CK, N1, N2 nitrogen application rates were respectively:0, 55.2, 110.4 kg/hm²) on the composition and structure of endophytic fungi community in different functional roots of millet. The results showed that the number of OTUs and fungal diversity (shannon index and Chao 1 index) of absorbing roots in CK treatment were significantly higher than those of transporting roots. At the phylum level, the abundance of Ascomycota and Glomeromycota of absorbing roots was significantly higher than that of transporting roots, but the abundance of Mortierellomycota and Basidiomycota was significantly lower than that of transporting roots. At the genus level, the relative abundance of the dominant genus Mortierella and Bipolaris in absorbing roots was significantly lower than that in transporting roots, but Fusarium and Minimedusa were significantly higher than transporting roots. The number of OTUs and Chao 1 index of absorbing root and transport root of high nitrogen fertilization increased by 23.62%, 23.87%, respectively, while other fertilization was just the opposite. The Shannon index of fertilization and transporting roots increased by 7.12%~19.62%, but the absorbing roots were the opposite. The relative abundance of fertilization-absorbing root pathogenic bacteria (Bipolaris, Fusarium, Magnaporthiopsis, Microdochium) increased by 52.99%, 40.74%, 133.06%, 200.00%, respectively, and the relative abundance of pathogen-resistant bacteria (Minimedusa) in transporting roots increased 40.16%~97.11%. The carbon, nitrogen, and phosphorus contents of the millet root system increased by 2.33%~11.63%, 13.30%~94.66%, 3.89%~263.92%, respectively. The effect of combined application of nitrogen and phosphorus was particularly significant. The biomass of fertilization and transport roots increased by 106.67%~336.41%, and the biomass of absorbing roots was reduced by 35.20%~60.20%. Long-term fertilization increased the abundance of endophytic fungi in absorbing roots by increasing soil available phosphorus, total phosphorus, soluble nitrogen and root carbon content, and reducing the abundance of endophytic fungi in transporting roots by increasing root biomass, and applying phosphorus fertilizer with low nitrogen (N1P) has the greatest impact on the soil nutrient content and the development of the flora. In short, long-term nitrogen application could change root endophytic flora by affecting the soil or root chemical composition, which might lead to the changes in plant survival strategies and ultimately affect farmland ecosystems.