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Gut-associated microbes of many insects provide a variety of beneficial nutritive functions to their hosts such as the provisioning of essential amino acids (EAAs) to those that feed on diets limited in assimilable nitrogen (i.e., wood). We investigated this function by the gut microbiota of the eastern subterranean termite (Reticulitermes flavipes) using 13C-stable isotope analysis of EAAs in the diet and termite samples. Evidence of possible microbe input was revealed by 13C-depletion of termite carcass (-27.0 ± 0.43‰, mean ± s.e.), and termite gut filtrate samples (-27.3 ± 0.58‰) relative to their wood diet (-26.0 ± 0.48‰) (F (2, 63) = 6.2, P < 0.004). An investigation of the identity of non-dietary EAA sources determined that termites predominantly incorporated EAAs derived from bacteria, with minor fungal input. The most likely means of EAA acquisition is through proctodeal trophallaxis (mouth-anus feeding), a well-established feature of termite colony nestmates, and subsequent digestion of the microbial fraction in the transferred food. Our study provides empirical data in support of the gut microbial EAA provisioning function in termites by using 13C-stable isotopes to determine the microbial origins of incorporated EAAs in termite tissues.