Characterization and in vitro inhibition studies of protease inhibitor from the mushroom Pleurotus floridanus (PfTI) towards the pest Papilio demoleus is studied. The addition of 1 mM Mn2+ , Na2+ , Ba2+ and Ni2+ enhanced the PfTI activity. The ICP-atomic emission spectrum showed the presence of Ca2+ , Mg 2+ and Zn 2+ in the PfTI. Surfactants SDS and CTAB at a concentration of 1% reduced the PfTI activity whereas, the nonionic detergents Triton X and Tween 80 increased the activity. The inhibitory activity gradually decreased with increase in concentration of DMSO and H2 O 2 . The activity was increased by dithiothreitol up to a concentration of 80 µM and inactivated at 140 µM. The activity of PMSF modified PfTI was drastically reduced to 0.234 U/mL at 4 mM concentration and similar results were obtained for modification of cysteine by N-Ethylmaleimide at slightly higher concentrations. The complex of trypsin and PfTI showed complete loss in fluorescence intensity at 343 nm compared with control. In vitro inhibition studies of PfTI with midgut proteases isolated from citrus pest P. demoleus with protease activity of 1.236 U was decreased to 0.613 U by 50 µL (0.1 mg/mL) of the inhibitor. Inhibitor was stable up to 0.04 M concentration of HCl.