Plutella xylostella (L.) (Lepidoptera: Plutellidae) is one of the most destructive pests of Brassicaceae vegetables. Cantharidin is an insect-derived defensive toxin, which has been reported to have toxicity to a variety of pests and especially lepidopteran pests. Although the toxicity of cantharidin on P. xylostella has been demonstrated, there is little information available on the specific detoxification response of P. xylostella against cantharidin. This study investigates the enzymatic response (including serine/threonine phosphatases [PSPs], carboxylesterases [CarEs], glutathione-S-transferases [GSTs], and cytochrome P450 monooxygenases [P450]) in P. xylostella to the sublethal and low lethal concentrations of cantharidin (LC₁₀ and LC₂₅). Results showed that the inhibitory activity of PSPs was increased and then decreased in vivo, while PSPs activity could be almost completely inhibited in vitro. Interestingly, the activities of detoxification enzymes (GST, CarE, and P450) in P. xylostella displayed a trend of decreasing and then increasing after exposure to the two concentrations of cantharidin. Notably, the increase in P450 enzyme activity was the most significant. The increasing trend of detoxification enzyme activity was congruent with the recovery trend of PSPs activity. This study contributes to our understanding of the detoxification mechanism of cantharidin in P. xylostella and helps in the further development of biogenic agents.