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Rotation and fertilization are important methods used to improve crop yield. In particular, crop rotation is an effective means of enhancing ecosystem diversity; however, there exist relatively few studies regarding the effects of long-term maize–soybean rotation and fertilization on soil microbial communities. To further understand the changes in soil microbial community structure under long-term maize–soybean rotation and fertilization, we used a 9-year-old experimental site with maize–soybean rotation as the research object and soybean continuous cropping as a control. We explored the growth effects of soybean and the changes in soil microbial communities under the soybean–maize rotation system and fertilization treatments by analyzing the physicochemical properties of the soil, crop agronomic traits, yield, and changes in soil microbial community structure. The results show that, in comparison with soybean continuous cropping, the yield of soybeans was increased by 12.11% and 21.42% under maize–soybean rotation with different fertilization treatments, respectively. Additionally, there was a significant increase in the agronomic effects of nitrogen following rotation combined with fertilization. Moreover, the soil pH, SOM, and nutrient status were also improved. Bryobacter, Gemmatimonas, and Rhodanobacter were the dominant bacteria. Rotation treatment increased the relative abundance of Bryobacter and Rhodanobacter, and fertilization treatment increased the relative abundance of Gemmatimonas. Rotation also increased the stability of the bacterial community structure and strengthened the symbiotic relationship between species. The prediction of nitrogen-related functional genes indicates that rotation increased soil ammonification and nitrification. Heterocephalacria and Mrakia were the dominant fungal genera under crop rotation. The abundance of Saccharomyces Mrakia was significantly positively correlated with ammonium nitrogen levels and crop yield. Crop rotation increased the abundance of Saccharomyces Mrakia and reduced the abundance of Fusarium, but fertilization increased the abundance of Fusarium. Functional gene prediction also indicates that the relative abundance of plant pathogens was significantly reduced. This study provides a theoretical basis for soil microbial diversity and ecosystem service function in long-term soybean–maize rotation. [ABSTRACT FROM AUTHOR]