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Crop rotation is a common farmland management measure, which can effectively improve soil permeability and soil fertility, reduce crop diseases and pests, and affect microbial activity and community structure to a certain extent. However, the effects of long-term crop rotation pattern on nitrogen cycle microorganisms and crop yield are still unclear. Using monocrop rice as a control (CK), this study looked at the effects of different rotation years of tobacco rice (rotation years 10–20 (T10), 20–40 (T20), and >40 years of rotation) on nitrogen cycling microorganisms, soil nitrogen content, and crop yield. The results showed that compared with CK, crop rotation mode improved soil fertility and increased soil nitrogen content. Among them, the ammonium nitrogen content of farmland under T20 treatment (increased by 32.27 % compared with CK), and the yields of tobacco and rice were the highest (increased by 43.12 % compared with CK; Compared with T10 and T40 treatment, the tobacco content was increased by 3.59 % and 1.82 %, respectively). In addition, crop rotation also changed the structure and diversity of nitrogen-cycling microorganisms. This study found that Proteobacteria contributed significantly to the increase of soil nitrogen content, and Geobacter , Nitrosospira , Frankia , and Burkholderia Cupriavidus were the key groups to increase soil nitrogen content and crop yield. Therefore, tobacco rice rotation increases soil fertility and crop yield, but the effect decreases after >40 years. This study provides a reference for ensuring soil nitrogen transformation and sustainable agricultural production. The research results laid a solid theoretical foundation for the efficient transformation and utilization of soil nitrogen and grain increase. [ABSTRACT FROM AUTHOR]