Abstract: In order to reveal the insecticidal activity and mechanism of podophyllotoxin, its bioactivity against Plutella xylostella larvae and its effect on their metabolic enzymes were assayed. The results showed that podophyllotoxin had high antifeedant and toxic activity, as well as obvious growth inhibitor activity against the moth. In 24-hour and 48-hour treatments, its AFC₅₀ were 0.4110 mg/mL and 0.2617 mg/mL, respectively. Its LC₅₀ of 72 h was 1.9061 mg/mL. When the tested insects were treated at the dosage of 0.125 mg/mL, their related growth rate reduced by 57.56% (24 h) and 41.18% (48 h) compared with the control; the pupation rate was only 43.33%, and the pupae did not eclose normally. At the same time, podophyllotoxin had different influence on metabolic enzymes. The activity of carboxyl esterases (CarE) in the tested larvae was first activated, and then inhibited. The activity of acid phosphatase (ACP) was first inhibited, and then activated. The activity of alkaline phosphatase (AKP) was inhibited obviously, and the inhibition became more intensive as the exposure time prolonged. Both glutathione S-transferase (GSTs) and cytochrome P450 were first induced, and then inhibited.