This work investigates the effect of methyl jasmonte (MeJa), mechanical wounding, and herbivory caused by larval feeding of a specialist insect ( Agraulis vanillae vanillae) upon trypsin inhibitory activity in passion fruit leaves. Despite the fact that all treatments caused accumulation of trypsin inhibitors (TIs), higher levels were observed in MeJa treated leaves when plants were assayed 24 and 48 h after stimulus. Concerning both mechanically injured plants and attacked ones, a systemic induction was observed. Partially purified inhibitors from MeJa exposed plants were further characterized by X-ray film contact print technique and N-terminal sequence. Such analysis indicated that the TIs identified belong to the Kunitz family. Moreover, the partially purified inhibitors strongly inhibited trypsin-like digestive enzymes from sugar cane stalk borer ( Diatraea saccharalis) in vitro. Our results further support the protective function of wound-inducible trypsin inhibitors and their potential as tools to improve important crop species against insect predation through genetic engineering.