Acetylcholinesterase (AChE) plays a pivotal role in the synaptic transmission in the cholinergic nervous system of most animals, including insects. Insects have two different ace (ace1 and ace2) loci that encode two distinct AChEs (AChE1 and AChE2), which were originated by duplication events long before the radiation of insects. However, little is known about when the ace duplication occurred and how each duplicated ace locus has evolved to retain the original functions. In this study, we conducted phylogenetic analysis for cholinesterase genes from all the lower animals with their genome sequenced together with all known arthropod ace1 and ace2, including those from a number of insects that were newly cloned. Among several independent duplications in lower animal lineages, one duplication event found in platyhelminthes appeared to be the direct origin of arthropod ace1 and ace2. Comparison of the evolutionary distance (d) of two aces from different insect groups relative to those from common ancestors revealed that ace1 has evolved with a significantly slower rate compared to ace2, suggesting that the ace1 lineage has maintained relatively more essential functions following duplication. When the dN/dS ratio was compared between ace1 and ace2 within different insect orders, ace2 was determined to have received relatively more positive selection pressure in Diptera and Hymenoptera whereas the same was true for ace1 in Coleoptera, Hemiptera and Lepidoptera. Along with the relatively more decreased d value for ace2, such an increased dN/dS ratio for ace2 in Diptera and Hymenoptera implied the incidence of functional transition of ace1 to ace2. Our findings should provide with new insights into the evolution of two insect AChEs: when they were generated and how they retain and gain the neuronal functions.