Fungal activity is a major driver in the global nitrogen cycle, and mounting evidence suggests that fungal denitrification activity contributes significantly to soil emissions of the greenhouse gas nitrous oxide (N 2 O). The metabolic pathway and oxygen requirement for fungal denitrification are different from those for bacterial denitrification. We hypothesized that the soil N 2 O emission from fungi is formate and O 2 dependent and that land use and landforms could influence the proportion of N 2 O coming from fungi. Using substrate-induced respiration inhibition under anaerobic and aerobic conditions in combination with 15 N gas analysis, we found that formate and hypoxia (versus anaerobiosis) were essential for the fungal reduction of 15 N-labeled nitrate to 15 N 2 O. As much as 65% of soil-emitted N 2 O was attributable to fungi; however, this was found only in soils from water-accumulating landforms. From these results, we hypothesize that plant root exudates could affect N 2 O production from fungi via the proposed formate-dependent pathway.