The chloro-nicotinyl insecticide imidacloprid is used extensively as a soil treatment against subterranean termites. We conducted the first study of the metabolic fate of imidacloprid in termites, by exposing workers of the eastern subterranean termite, Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae) to radiolabeled imidacloprid through topical application and ingestion. Several days after topical application, we detected up to 11 radiolabeled compounds. The parent compound, IMI, and the following six metabolites were identified by liquid chromatography/mass spectrometry: olefin-imidacloprid (major metabolite), 4/5-OH imidacloprid, 4,5-di-OH imidacloprid, des-nitro olefin imidacloprid, des-nitro imidacloprid, and a glucuronide conjugate of des-nitro imidacloprid (des-nitro IMI-glu). Over time, detoxification of imidacloprid proceeded from less polar to more polar compounds, with des-nitro IMI-glu seeming to be the ultimate, major end product in surviving termites. Degradation of imidacloprid was limited to internal tissues of the termite. Workers fed wood treated with imidacloprid or provided with a treated substrate (sand) had metabolitic profiles similar to topically treated termites. Termites fed imidacloprid or exposed to it in soil excreted detectable amounts of all of the identified metabolites. Finally, we determined that imidacloprid metabolites were less toxic to termites than imidacloprid itself. Only the olefin-imidacloprid and 5-OH imidacloprid caused morbidity in termites exposed to sand treated with these compounds, but at concentrations ≈10–20-fold higher than the parent imidacloprid. Our results shed light on the metabolic pathway used by termites to detoxify imidacloprid and show how metabolism plays a key role in determining the availability of the active ingredient and its various metabolites for transfer among workers within the colony.