In this study, the insecticidal active substance of the fruiting body of Paecilomyces cicadae (Miquel) Samson that can kill the larvae of Plutella xylostella (Lepidoptera: Plutellidae) was isolated and purified by combined separation with biology experiments for the first time. By means of bioactive guided isolation of 40% ethanol-H2O, dichloromethane, ethyl acetate,n-butyl ethanol, macroporous adsorption resin column, high performance liquid chromatography, and sephadex A-20 column, the insecticidal active compound was purified,and a white powder compound was obtained. The compound was active against the third instar larvae of P. xylostella in 24, 48, and 72 h at the concentration of 10.0 mg mL-1 with a corrective mortality of (95.1 ± 1.8), (97.7 ± 1.6), and (99.1 ± 1.7)%, respectively. The compound was strongly sensitive to the larvae. It was stable at acid environment, and was not sensitive to pepsin, and other chracteristics were studied.