Camptothecin (CPT) is a prominent molecule in natural product research because of its application prospects in medicine and agriculture. In this study, CPT and its derivatives were discovered to be competitive inhibitors of group II and group h insect chitinases, both of which are key components of insect chitinolytic systems. CPT and 7-ethyl-10-hydroxycamptothecin (SN-38) inhibited group II chitinase from Ostrinia furnacalis (OfChtII) with K(i) values of 5.1 and 2.0 muM, respectively. Results from tryptophan fluorescence spectroscopy, molecular docking analysis, and molecular dynamics simulations revealed that both CPT and SN-38 inhibit OfChtII-C1 by interacting with solvent-exposed tryptophan residues in a substrate-binding cleft. CPT exhibited high insecticidal activity toward the orthopteran pest Locusta migratoria, possibly because of the midgut metabolism of CPT, with only moderate activities toward lepidopteran pests. Even though SN-38 exhibited much lower insecticidal activities than CPT, it still showed higher inhibitory activity toward chitinase. This study reports a new molecular target of CPT and provides insights into molecular design of CPT-based insecticides against different kinds of pests.