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Acetylcholinesterase (AChE) regulates nerve signalling and is a well-validated target for insect control in both agriculture and the prevention of mosquito-borne diseases. However, current AChE-targeting insecticides are nonspecific and thus also affect other organisms such as honey bees. The synaptic AChE of honey bees (AChE2) is encoded by the ace-2 gene, thought to have originated from a gene duplication of ace-1. Here, we analyse the structure, dynamics, and kinetics of AChE2 enzymes from the honey bee, Apis mellifera (AmAChE2), and the malaria mosquito, Anopheles gambiae (AgAChE2), and compare them to the more extensively studied type 1 mosquito AChE (AgAChE1) and mammalian AChEs. Important differences between these AChE subtypes were identified. Profiling with selected noncovalent AChE inhibitors revealed strong AChE2 inhibitors, but the inhibition profiles of AChE2 differed substantially from those for AgAChE1 and human AChE. Modelling of AChE2•inhibitor complexes revealed two tyrosines unique to AChE2 that are responsible for these differences in inhibitor sensitivity. These results highlight the importance of considering molecular properties of AChE2 when developing AChE1 inhibitors for pest control. Furthermore, the results also suggest that including AChE2 in computer-aided molecular design efforts during the discovery process could be very valuable for reducing risks of off-target effects.