Crop rotation is recognized as a sustainable practice, yet the microbial mechanisms underlying its benefits in subtropical rice systems remain unclear. To explore how rotation is linked to soil microbial communities and agroecosystem functioning, we surveyed microbial communities, functional genes, and environmental factors across 45 rice paddies in the Anning River Valley using sequencing and metagenomics. Compared with rice monoculture (RM), rotation systems (RR) increased yield by 17.18 %, along with distinct microbial composition, stronger deterministic assembly, and more modular ecological networks. Rare taxa showed higher Shannon diversity than abundant taxa and were more responsive to environmental variables, particularly soil organic carbon (SOC), which accounted for up to 42 % of their variation. Microbial networks in RR soils exhibited greater topological stability and keystone taxa, including abundant Proteobacteria and rare Ascomycota, were positively correlated with yield. Genes involved in carbon oxidation (fdoG), nitrogen fixation (nifH), and phosphorus mineralization (phnP) also showed positive yield associations. Structural equation modeling identified significant links among soil moisture, SOC, and yield, suggesting microbial mediation. Notably, although yield increased, nutrient-cycling multifunctionality declined at 86 % of sites, indicating a trade-off. However, a moderate positive correlation (R² = 0.214) between multifunctionality and yield suggested that trade-offs were not fixed and that dual improvement was possible with targeted management. These findings indicate that crop rotation is associated with a restructured soil microbiome characterized by altered keystone taxa, nutrient-cycling genes, and a shift toward more deterministic assembly, providing a framework for designing subtropical rice systems that integrate productivity with ecological sustainability.