AbstractProteases present in the salivary glands of phytosuccivorous insects are responsible for the initial breakdown of dietary protein into the amino acids necessary for insect growth and development. Here, we characterised the total proteases and trypsin‐like activities in the salivary glands of the Neotropical brown stink bug,Euschistus heros(F.) and used in silico and biochemical approaches to determine the interactions between trypsin‐like enzymes and the soybean Kunitz trypsin inhibitor (SKTI). Additionally, we evaluated whether these enzyme activities were affected in individuals resistant to the actions of the insecticide imidacloprid. We used an originally field‐collected strain to select individuals resistant to imidacloprid alone (ImiGoiasRes) or synergised with piperonyl butoxide (ImiPBORes). A laboratory‐susceptible (ImiSusc) strain was also used to select piperonyl butoxide synergised imidacloprid‐resistant (ImiPBOSel) individuals. Our results revealed that pH and temperature significantly affected the specific trypsin‐like activities in imidacloprid‐susceptible individuals. Significant alterations in the total protease activities were recorded for field‐ (ImiGoiasRes) and laboratory‐selected imidacloprid‐resistant individuals (ImiPBOSelandImiPBORes). Our computational docking analysis demonstrated that the molecular interactions and the presence of the SKTI inhibitor (0.5 mg mL−1) could reduce up to 40% of the trypsin‐like activities of imidacloprid‐susceptible individuals. However, all imidacloprid‐resistant strains exhibited significantly less trypsin‐like activities and exhibited lower hydrolysing velocity (VMAX) and affinity (KM) rates, and the prior exposure to SKTI only synergised the imidacloprid toxicity for individuals ofImiFieldResstrain. Collectively, our findings demonstrate, for the first time, insecticide resistance detrimentally affected the activities of proteolytic enzymes in phytosuccivorous stink bug pests.