Xylophagous termites possess symbiotic bacteria that fix atmospheric nitrogen (N₂). Although symbiotic N₂ fixation is central to termite nutrition and ecologically important, it is energetically costly. Using stable isotopes, we tested the hypothesis that symbiotic N₂ fixation would decrease in workers of the eastern subterranean termite, Reticulitermes flavipes Kollar, which were exposed to high nitrogen diets. To calculate the isotope discrimination factor occurring as a result of digestion, Δ_dig, and which occurs as the result of N₂ fixation, Δ_fix, symbiotic N₂ fixation was inhibited via force feeding termites the antibiotic kanamycin. Antibiotic-treated termites and control (N_2-fixing) termites were exposed to different concentrations of dietary N (0, 0.21, and 0.94% N), their ¹⁵N signatures were obtained, and the percent nitrogen derived from the atmosphere within termite samples was calculated. As we hypothesized, symbiotic N₂ fixation rates were negatively correlated with dietary N, suggesting that high concentrations of dietary N suppressed symbiotic N₂ fixation in R. flavipes. A comparison of the ¹⁵N isotope signatures of antibiotic-treated termites with their food sources demonstrated that Δ_dig = 2.284‰, and a comparison of the ¹⁵N isotope signatures of antibiotic-treated termites with control termites indicated that Δfix = -1.238‰. These are the first estimates of Δ_dig for R. flavipes, and the first estimate of Δ_fix for any N₂-fixing termite species.