Abstract: In order to investigate the insecticidal activity and mechanism of matrine, oxymatrine and kaempferol on 3rd instar larvae of Lymantria dispar, the effects of three plant secondary metabolites on the survival and three enzymes (GST, AChE and CarE) of L. dispar were analyzed by bioassay, in vitro and in vivo enzyme activity assay. The results indicated that matrine, oxymatrine and kaempferol all possessed a highly insecticidal activity on 3rd instar larvae of L. dispar, and matrine showed the highest insecticidal activity among three plant secondary metabolites with a LC₅₀ value of 0.420 mg/mL. In vitro enzyme activity assays showed that the activities of GST, AChE and CarE could be inhibited by three plant secondary metabolites. The IC₅₀ values of oxymatrine against GST and AChE were 0.928 and 0.717 mg/mL, respectively, while the IC₅₀ value of matrine against CarE was 0.436 mg/mL, which was the highest inhibition. Furthermore, the results of in vivo enzyme activity assay indicated that matrine had the highest inhibition on GST, which showed the lowest activity at 48 hours after treatment and was 39% of the control at the same stage. Moreover, oxymatrine had the highest inhibition on AChE and CarE, and their activities were 44% and 40% of the control at 72 hours after treatment, respectively.The above results indicate that the inhibition of matrine, oxymatrine and kaempferol on three enzymes (GST, AChE and CarE) of L. dispar is one of the reasons for their insecticidal activity.