BACKGROUND: In insects, carboxylesterases (CarEs) are enzymes involved in the detoxification of insecticides. However, the molecular mechanism of CarE-mediated insecticide metabolism in Bradysia odoriphaga, a serious agricultural pest, remains unclear. The aim of this study is to investigate the detoxification process of malathion, bifenthrin, and imidacloprid by B. odoriphaga carboxylesterase (Boest1).RESULTS: An alpha class CarE gene Boest1 was cloned from B. odoriphaga. The results of real-time quantitative polymerase chain reaction showed that Boest1 is up-regulated with age during the larval stage, and the level of transcription of Boest1 is higher in the midgut and Malpighian tubule than in other tissues. The expression level of Boest1 was significantly increased after exposure to malathion and bifenthrin. Recombinant BoEST1 expressed in vitro showed high catalytic activity toward ⊍-naphthyl acetate, which was substantially inhibited by malathion and triphenyl phosphate. The in vitro metabolism assays showed that BoEST1 demonstrates hydrolytic capacity toward malathion and bifenthrin but not imidacloprid. The binding free energy analysis indicates that BoEST1 has a higher affinity for malathion and bifenthrin than imidacloprid.CONCLUSION: These results suggest that BoEST1 plays a role in the breakdown of insecticides and may be involved in the development of resistance in the Chinese chive pest B. odoriphaga; our findings also provide data for better pest management and perspectives for new pesticides development.