Resistance to insecticides and acaricides is a major impediment to effectively controlling insect pests worldwide. These pests include the two-spotted spider mite Tetranychus urticae (T. urticae), which exists globally. This polyphagous herbivore causes major agricultural problems and can develop resistance to the agents above. Therefore, the continuous development of acaricides with new modes of action is important to circumvent the resistance of insects to pesticides. Acynonapyr is a novel class of acaricides containing an azabicyclo ring. In this study, we determined the activity of acynonapyr and its analogs on calcium-activated potassium (K Ca 2) channels in two-spotted spider mites using electrophysiological techniques (patch-clamp). We also examined their acaricidal efficacy against mites in the laboratory. The acynonapyr and analogs blocked T. urticae K Ca 2 (TurK Ca 2) channels in a concentration-dependent manner. A comparison of acaricidal activity against T. urticae with inhibitory activity against TurK Ca 2 revealed that TurK Ca 2 channels are the primary toxicological targets. Finally, we examined the effect of acynonapyr on Homo sapiens K Ca 2 (HsaK Ca 2.2) channels and demonstrated that the compound at 10 μM had a limited effect on the activity of this channel.