Disturbances have long been recognized as important forces for structuring natural communities but their effects on trophic structure are not well understood, particularly in terrestrial systems. This is in part because quantifying trophic linkages is a challenge, especially for small organisms with cryptic feeding behaviors such as insects, and often relies on conducting labor-intensive feeding trials or extensive observations in the field. In this study, we used stable isotopes of carbon and nitrogen to examine how disturbance (annual biomass harvesting) in tallgrass prairies affected the trophic position, trophic range, and niche space of ants, a widespread grassland consumer. We hypothesized that biomass harvest would remove important food and nesting resources of insects thus affecting ant feeding relationships and trophic structure. We found shifts in the feeding relationships inferred by isotopic signatures with harvest. In particular, these shifts suggest that ants within harvest sites utilized resources at lower trophic levels (possibly plant-based resources or herbivores), expanded trophic breadth, and occupied different niche spaces. Shifts in resource use following harvest could be due to harvest-mediated changes in both the plant and arthropod communities that might affect the strength of competition or alter plant nitrogen availability. Because shifts in resource use alters the flow of nutrients across the food web, disturbance effects on ants could have ecosystem-level consequences through nutrient cycling.