The effect of large herbivores on gaseous N loss from grasslands, particularly via denitrification, is poorly understood. In this study, we examined the influence of native migratory ungulates on denitrification in grasslands of Yellowstone National Park in two ways, by (1) examining the effect of artificial urine application on denitrification, and (2) comparing rates inside and outside long-term exclosures at topographically diverse locations. Artificial urine did not influence denitrification 3 and 12 days after application at hilltop, mid-slope, and slope-bottom sites. Likewise, grazers had no effect on community-level denitrification at dry exclosure sites, where rates were low. At mesic sites, however, ungulates enhanced denitrification by as much as 4 kg N ha−1 year−1, which was double atmospheric N inputs to this ecosystem. Denitrification enzyme activity (DEA, a measure of denitrification potential) was positively associated with soil moisture at exclosure sites, and herbivores stimulated DEA when accounting for the soil moisture effect. Glucose additons to soils increased denitrification and nitrate additions had no influence, suggesting that denitrification was limited by the amount of labile soil carbon, which previously has been shown to be enhanced by ungulates in Yellowstone. These results indicate that denitrification can be an ecologically important flux in portions of semi-arid landscapes, and that there is a previously unsuspected regulation of this process by herbivores.