Termites are remarkable for their ability to digest cellulose from wood as their main energy source, but the extremely low nitrogen (N) content of their diet presents a major challenge for N acquisition. Besides the activity of N 2 -fixing bacteria in the gut, the recycling of N from waste products by symbiotic microbes as a complementary N-provisioning mechanism in termites remains poorly understood. In this study, we used a combination of high-throughput amplicon sequencing, quantitative PCR, and cultivation to characterize the microbial community capable of degrading urea, a common waste product, into ammonia in the guts of termites ( Reticulitermes hesperus ) from a wild and laboratory-reared colony. Taxonomic analysis indicated that a majority of the urease ( ureC ) genes in the termite gut (53.0%) matched with a Treponema endosymbiont of gut protists previously found in several other termites, suggesting an important contribution to the nutrition of essential cellulolytic protists. Furthermore, analysis of both the 16S rRNA and ureC amplicons revealed that the laboratory colony had decreased diversity and altered community composition for both prokaryotic and ureolytic microbial communities in the termite gut. Estimation by quantitative PCR showed that microbial ureC genes decreased in abundance in the laboratory-reared colony compared to the wild colony. In addition, most of our cultivated isolates appeared to originate from non-gut environments. Together, our results underscore a more important role for ureolysis by endosymbionts within protists than by free-swimming bacteria in the gut lumen of R. hesperus .