Objective The object of this study was to explore the effect of nitrogen addition rate on the relationship between soil microbial diversity and asynchrony in rice-wheat rotation.Method We collected soil samples at key growth stages in rice-wheat rotation under different nitrogen addition rates in a long-term experiment and used high-throughput sequencing technology to analyze the effect of nitrogen addition rate on soil microbial diversity. Also, we explored the effects of nitrogen addition rates on yield by altering soil microbial asynchrony. The gradient N addition rates in field experiment were 0, 50, 100, 200, 300 kg·hm^–2 for wheat and 0, 90, 180, 270, 360 kg·hm^–2 for rice. The key growth stages when soil samples were collected include: fallow before wheat planting, jointing, booting, flowering, and maturing during the wheat season, and fallow before rice planting, max-tillering, shooting, flowering, and maturing during rice season.Result N addition rates impacted soil microbial diversity and composition in each growth stage, and the N addition rate could significantly account for about 12% variations of microbial richness in the rice period. When the N addition rates were 100 or 180 kg·hm^–2, soil microbial diversity in the wheat or rice seasons, respectively, was maintained at a relatively high level across all plant developmental stages. Also, the N addition rate could significantly account for 9%-11% variations in microbial community composition in the wheat and rice period. With the increase of N addition rate, the asynchrony of some microbial populations was significantly increased during the wheat (e.g. Phenylobacterium, Sphingomonas, Cyanobacteria GpI, Desulfovirga, Lacibacter, Terrimonas) and rice seasons (e.g. Desulfovirga, Spartobacteria genera incertae sedis, Ohtaekwangia, Acidobacteria Gp7, Arenimonas, Niastella). Importantly, the wheat and rice yields showed positive relationships with the asynchronies of Phenylobacterium and Desulfovirga and with the asynchronies of Desulfovirga, Spartobacteria genera incertae sedis, Ohtaekwangia and Arenimonas, respectively.Conclusion Nitrogen addition rate has a constant impact on soil microbial diversity in rice-wheat rotation during plant development, which changes microbial population asynchrony, and then improves certain functional complementation to increase crop yield. The results of this study can provide a scientific basis and practical guidance for regulating soil microbial communities to maintain high crop yield.