To understand paddy-upland rotation ecosystem microbial mechanisms, the community composition and assembly processes in a paddy-upland rotation ecosystem were investigated. In addition, the samples collected from long-term flooded lotus field ecosystem and fruit-planted upland ecosystem with the same parent material were also evaluated as the control. Results showed that due to the particular niche pattern driven by frequent farming activities and fertilization management, deterministic processes dominated microbial community assembly in both upland and paddy-upland ecosystems. In upland, the deterministic processes were up to 78.6%, while stochastic processes were 10.7%. With the intermittent flooding, paddy-upland ecosystem undergoes a relatively high soil fertility accumulation and favorable hydrologic connectivity, which formed a gradual-changed inner condition. Compared to upland ecosystem, the stochastic processes (39.3%) increased in paddy-upland ecosystem. In the same way, the stochastic processes (50.0%) dominated community assembly in long-term flooded ecosystem. In total, microbial assembly processes exert great influences on community composition and functions, the increased stochastic processes favor the microbial diversity and the interactions among microbes, enhance community resistance towards disturbance, which in turn benefit the stability and sustainability of agroecosystem functions.