The study of changes that occurred in insect organisms in response to insecticidal exposure and their species-specific characteristics is important for a fuller understanding of the environmental and evolutionary patterns of pesticidal resistance. For chlorfenapyr from the pyrrole group of insecticides, the mechanism underlying the resistance in insects is not quite clearly described. This study evaluated the activity of acetylcholinesterase (AChE) in adults of the house fly Musca domestica of the chlorfenapyrresistant (selected with chlorfenapyr) strain (ChlA). Also, we assessed the kinetic parameters of AChE in females and males of the ChlA strain compared to these of the unselected strain (Lab) of M. domestica for the first time. Specimens of Lab and ChlA strains had no statistically significant differences in specific AChE activity. The percentage remaining activities of propoxur-inhibited AChE was 3.81 times less (р < 0.05) and values of Vmax and Km were 43.3% and 46.9% (р < 0.05), respectively, less in females of the ChlA strain compared to these in females of the Lab strain. For both Lab and ChlA strains M. domestica, the catalytic efficacy of AChE based on Vmax/Km in males was more than that in females. In general, the results obtained suggest that the affinity of AChE to specific ligands (like a substrate acetylthiocholine and an inhibitor propoxur) increased without a rise of the catalytic activity in females of the ChlA strain M. domestica that was under selection with chlorfenapyr during 23-24 generations.