From the methanol crude extracts of the tree of heaven (Ailanthus altissima) leaves, the antifeedant substance was isolated and bioassayed with different concentrations against diamondback moth (Plutella xylostella) larvae. The antifeeding activity was evaluated by measuring the feeding area during 24 hr after inoculation. Methanol extracts showing antifeeding activity at 5000 ppm was subsequently fractionated into hexane, chloroform, ethyl acetate and water layer. Third larvae of diamondback moth was tested to each fraction layer. Chloroform layer shows the highest antifeeding activity and the layer was purified by silica gel open column chromatography. The C22 and C23 fractions showed higher antifeeding ratio with 96 and 86%, respectively, and then these two fractions were re-isolated by ODS open column chromatography. As a result, both fractions in methanol 40% (v/v) showed antifeeding ratio over 90%. The C221 fraction showed insecticidal activity in all fraction, however, C231 fraction was showed the antifeeding activity only in C2311 fraction. The C2311 fraction judging to have antifeeding activity was re-isolated and purified by HPLC and recycling, and finally obtained the bioactive substances (C23111) with antifeeding ratio with 88%. The structure of bioactive materials isolated was confirmed by LC-mass and 1H-NMR(500 MHz), 13C-NMR(100 MHz).