The molecular and biochemical properties of two acetylcholinesterases (LdAChE1 and LdAChE2) from the Colorado potato beetle, Leptinotarsa decemlineata, were investigated in this study. Polyacrylamide gel electrophoresis in conjunction with western blotting with LdAChE1- or LdAChE2-specific antibodies suggested that LdAChE1 exists in a soluble form, whereas LdAChE2 exists in both soluble and amphiphilic forms with a glycophosphatidylinositol anchor. Both LdAChEs exist as homodimers with each monomer connected with a disulfide bond. LdAChE1 was the most highly expressed in the thorax followed by the head, leg, and abdomen, whereas LdAChE2 was the most highly expressed in the head, followed by the thorax, leg, and abdomen. The overall expression levels of LdAChE1, however, were higher than those of LdAChE2 in all examined tissues. Kinetic analysis using recombinant LdAChE1 and LdAChE2 showed that LdAChE2 has a 4.8-fold higher catalytic efficiency toward acetylthiocholine iodide compared to LdAChE1. LdAChE2 was more sensitive to organophosphorus and carbamate insecticides than LdAChE1. The addition of irreversibly phosphorylated LdAChE1 via paraoxon titration significantly reduced LdAChE2 inhibition by insecticides and glycoalkaloids, suggesting a sequestration role of soluble LdAChE1 in the chemical defense against xenobiotics. Taken together, LdAChE2 may be the main enzyme for synaptic transmission, thus serving as a toxicologically more relevant target, whereas the soluble LdAChE1 may function as a bioscavenger.