A novel 33-kD cysteine protease (Mir1-CP) accumulates at the feeding site in the whorls of maize (Zea mays L.) lines that are resistant to herbivory by fall armyworm (Spodoptera frugiperda) and other lepidopteran species. When larvae were reared on resistant plants, larval growth was reduced due to impaired nutrient utilization. Scanning electron microscopy (SEM) indicated that the peritrophic matrix (PM) was damaged when larvae fed on resistant plants or transgenic maize callus expressing Mir1-CP. To directly determine the effects of Mir1-CP on the PM, a baculovirus-based system was used to express the protease in Trichoplusia ni larvae. The expressed protein was collected from the hemolymph and purified by size exclusion and reverse phase chromatography. The effect of purified Mir1-CP on PM permeability was determined by measuring the movement of blue dextran across the PM. Mir1-CP increased the permeability of the PM in a concentration-dependent manner. Inclusion of E64, a specific cysteine protease inhibitor prevented the damage. Mir1-CP damaged the PM at pH values as high as 8.5 and was more active than equivalent concentrations of papain and bromelain. These results indicated that an inducible plant cysteine protease directly damages the PM in vitro.