N2O is a potent greenhouse gas involved in the destruction of the protective ozone layer in the stratosphere and contributing to global warming. The ecological processes regulating its emissions from soil are still poorly understood. Here, we show that the presence of arbuscular mycorrhizal fungi (AMF), a dominant group of soil fungi, which form symbiotic associations with the majority of land plants and which influence a range of important ecosystem functions, can induce a reduction in N2O emissions from soil. To test for a functional relationship between AMF and N2O emissions, we manipulated the abundance of AMF in two independent greenhouse experiments using two different approaches (sterilized and re-inoculated soil and non-mycorrhizal tomato mutants) and two different soils. N2O emissions were increased by 42 and 33% in microcosms with reduced AMF abundance compared to microcosms with a well-established AMF community, suggesting that AMF regulate N2O emissions. This could partly be explained by increased N immobilization into microbial or plant biomass, reduced concentrations of mineral soil N as a substrate for N2O emission and altered water relations. Moreover, the abundance of key genes responsible for N2O production (nirK) was negatively and for N2O consumption (nosZ) positively correlated to AMF abundance, indicating that the regulation of N2O emissions is transmitted by AMF-induced changes in the soil microbial community. Our results suggest that the disruption of the AMF symbiosis through intensification of agricultural practices may further contribute to increased N2O emissions.The ISME Journal advance online publication, 19 December 2013; doi:10.1038/ismej.2013.224.