Allocation of carbon (C) to root metabolism in grasslands is poorly understood, even though root metabolism is thought to make up a large percentage of a plant’s total C budget. Moreover, little is known about the impact of herbivory on grassland root metabolism, which may be significant, based on work indicating that grazers can substantially affect plant biomass allocation. The objective of this study was to examine the effect of defoliation on community-level root metabolism in grasslands of Yellowstone National Park. Root metabolism and biomass were measured in 2006 in control, ungrazed and grazed plots in three grassland communities that varied in elevation, topographic position, edaphic properties, and plant productivity. Defoliation either increased or decreased root metabolic rates per gram of root dependent upon site and time after grazing. However, across all three sites the greatest divergence between control and grazed plots occurred approximately 15 days following defoliation and at the end of the growing season. These results indicate that the response of grassland root metabolism to defoliation has both spatial and temporal components. Changes in plant C allocation to root metabolism by herbivores altered Yellowstone grassland C budgets, which, by inference, altered plant C flows to soil food webs.