To explore the impact of long-term application of pig manure on diazotrophic community, soils were sampled under 4 kinds of fertilizations: CK, without pig manure, and M9, M18, M27, application of 9, 18 and 27 Mg/hm² pig manure, respectively from the Yingtan Red Soil Ecology Experimental Station, Chinese Academy of Sciences. High-throughput sequencing was utilized to measure the diversity and community structure of diazotrophs. Results showed that long-term application of pig manure significantly increased the diversity of diazotrophs, which was positively correlated with soil organic carbon, total nitrogen, and available nutrients, but not significantly correlated with soil pH. Non-metric multidimensional scaling (NMDS) analysis indicated that, compared with CK, long-term pig manure application significantly altered the community structure of diazotrophs, and the degree of difference increased with the increasing application amount of pig manure. Mantel test analysis revealed that the community structure of diazotrophs was mainly affected by dissolved organic carbon (DOC), available potassium (AK) and phosphorus (AP). Long-term application of pig manure significantly increased the relative abundance of Cyanobacteria and Firmicutes, while decreased the relative abundance of α-Proteobacteria and β-Proteobacteria, which could be ascribed to the change of soil nutrients. In conclusion, long-term application of pig manure can significantly increase the diversity, and alters the community structure and composition of diazotrophs in an acidic Ultisol, while the alteration of soil nutrient contents might be the key driving factor.