In order to better understand the insecticidal activity and mode of action of spinosad，its effect on the activity of protective enzymes and chitinase of 3rd and 5th instar larvae of Lymantria dispar was assayed in this study. The results showed that spinosad had no effect on catalase (CAT) activity, but significantly changed phenoloxidase (PO) activity in an activationinhibition fashion for both 3rd and 5th instar larvae. The same significant “activationinhibition” effect was also found on the activity of superoxide dismutase (SOD) and peroxidase (POD) in the 3rd instar larvae (P001), with the maximum enzyme activations occurred 3-6 hours after spinosad treatment. Whereas the POD and SOD activity in the 5th instar larvae was significantly increased within 3 hours after the treatment (P001; with treatment/CK ratios being 1.543 and 1.716, respectively), and no significant treatment effects were detected after 3-6 hours. The chitinase activity in the 3rd instar larvae was significantly activated 3-12 hours after spinosad treatment, while it was strongly inhibited in the 5th instar larvae 12-24 hours after the treatment. In conclusion, spinosad shows a significant effect on the activity of most protective enzymes and chitinase, and can interrupt normal metabolism of L. dispar larvae, therefore, has a potential to be an effective pesticide against this serious defoliator pest.