Almost all insect species possess two different acetylcholinesterases (AChE1 and AChE2) but it still remains unknown which AChE plays a major role in synaptic transmission. To predict the evolutionary distributions of functional AChE, relative amount and activity of two AChEs were investigated by native-PAGE in conjunction with Western blotting using AChE1- and AChE2-specific antibodies. Among 39 insect species examined, AChE1 was expressed as the main enzyme in 26 insect species across diverse taxa, suggesting that AChE1 likely plays a more crucial role in these insects. In contrast, AChE2 was predominantly expressed in remaining insect species, including paleopteran insects, suggesting that the replacement of AChE1 function with AChE2 is an old event. As expected, only AChE2 was detected in Cyclorrhaphan flies, supporting the notion that AChE2 in Cyclorrhapha has completely replaced the physiological functions of AChE1 during the evolution of Diptera. Taken together, contrary to the common belief that AChE1 is the major enzyme in most insects, many insect species across various taxa employ AChE2 as the main AChE, suggesting that functional transition from AChE1 to AChE2 has occurred rather universally and randomly with multiple independent origins within the class Insecta. This finding should provide valuable insights into which AChE has evolved to undertake the synaptic function and how functional diversification of AChE has occurred.