The red flour beetle (Tribolium castaneum) is a global agricultural grain storage pest that seriously jeopardizes the food safety of wheat, corn, and other grains. Carboxylesterases belong to a family of multifunctional enzymes that can participate in the metabolism of toxic substances ingested by T. castaneum, conferring drug resistance. In this study we have cloned from T. castaneum a gene encoding carboxylesterase, α-EST5, which is expressed mainly in T. castaneum late larvae and late adults. Tissue expression pattern analysis showed that α-EST5 was mainly expressed in the larval gut and adult head. Moreover, α-EST5 was able to be induced to be expressed by mugwort essential oils and significantly increased the enzymatic activity of carboxylesterase in T. castaneum after mugwort essential oil treatment. The sensitivity of T. castaneum to mugwort essential oils was significantly increased after knockdown the expression of α-EST5 using RNAi (RNA interference) technology. Through our study, we demonstrated that the carboxylesterase α-EST5 plays an important role in the detoxification of mugwort essential oils by T. castaneum. This study results provide a reference for understanding the detoxification mechanism of T. castaneum to external toxic substances.