Mycorrhizae are ubiquitous symbiotic associations between arbuscular mycorrhizal fungi (AMF) and terrestrial plants, in which AMF receive photosynthates from and acquire soil nutrients for their host plants. Plant uptake of soil nitrogen (N) reduces N substrate for microbial processes that generate nitrous oxide (N2O), a potent greenhouse gas. However, the underlying microbial mechanisms remain poorly understood, particularly in agroecosystems with high reactive N inputs. We examined how plant roots and AMF affect N2O emissions, N2O-producing (nirK and nirS) and N2O-consuming (nosZ) microbes under normal and high N inputs in conventional (CONV) and organically managed (OM) soils. Here, we show that high N input increased soil N2O emissions and the ratio of nirK to nirS microbes. Roots and AMF did not affect the (nirK + nirS)/nosZ ratio but significantly reduced N2O emissions and the nirK/nirS ratio. They reduced the nirK/nirS ratio by reducing nirK-Rhodobacterales but increasing nirS-Rhodocyclales in the CONV soil while decreasing nirK-Burkholderiales but increasing nirS-Rhizobiales in the OM soil. Our results indicate that plant roots and AMF reduced N2O emission directly by reducing soil N and indirectly through shifting the community composition of N2O-producing microbes in N-enriched agroecosystems, suggesting that harnessing the rhizosphere microbiome through agricultural management might offer additional potential for N2O emission mitigation.