| Summary: | Soil bacteria are key components of the soil microbial community contributing to soil health. Nitrogen (N) fertilization is an important factor that affects soil microbial community and cereal production. This study aims to explore the impact of long-term N fertilization on soil bacterial diversity, nitrogen use efficiency (NUE), and the grain yield of wheat in the semiarid region of Loess Plateau, China. The field experiment was conducted from 2003 to 2018 including five N treatments: 0 (N0), 52.5 (N52.5), 105 (N105), 157.5 (N157.5), and 210 (N210) kg N ha<sup>−1</sup> yr<sup>−1</sup>. The soil pH was decreased by the N fertilization, while the soil ammonium, nitrate, and available phosphorus were increased. The N uptake and grain yield of wheat were significantly increased with N and the highest NUE (28%) and grain yield (44% higher than control) were observed at 105 kg N ha<sup>−1</sup>, but no significant increase in yield was observed by further increasing N rate. The bacterial diversity was significantly increased at N105. Soil bacteria community was strongly related to soil chemical properties and ammonium content was the most important contributor. The dominant soil bacterial phyla were Proteobacteria, Actinobacteria, Acidobacteria, Chloroflexi, Gemmatimonadetes, Bacteroidetes, Nitrospirae, Verrucomicrobia, and Planctomycetes. The higher grain yield of wheat was related to the higher class Gammaproteobacteria and Sphingobacteriia abundance, and lower class Acidobacteria and Chloroflexia abundance. In summary, 105 kg ha<sup>−1</sup> yr<sup>−1</sup> was the optimum rate of N for diversified soil bacterial community and wheat yield for sustainable wheat production in semiarid Loess Plateau of China, whose higher N use efficiency was attributed to the higher phyla Verrucomicrobia and Planctomycetes, and lower Proteobacteria abundance.
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