The carmine spider mite, Tetranychus cinnabarinus (Boisduval), is a major agriculture pest. It can be found worldwide, has an extensive host plant range, and has shown resistance to pesticides. Organophosphate and carbamate insecticides account for more than one-third of all insecticide sales. Insecticide resistance and the toxicity of organophosphate and carbamate insecticides to mammals have become a growing concern. Acetylcholinesterase (AChE) is the major targeted enzyme of organophosphate and carbamate insecticides. In this study, we fully cloned, sequenced and characterized the ace1 gene of T. cinnabarinus, and identified the differences between T. cinnabarinus AChE1, Tetranychus urticae Koch AChE1, and human AChE1. Resistance-associated target-site mutations were displayed by comparing the AChE amino acid sequences and their AChE three-dimensional (3D) structures of the insecticide-susceptible strains of T. cinnabarinus and T. urticae to that of a T. urticae-resistant strain. We identified variation in the active-site gorge and the sites interacting with gorge residues by comparing AChE1 3D structures of T. cinnabarinus, T. urticae, and humans, though their 3D structures were similar. Furthermore, the expression profile of T. cinnabarinus AChE, at the different developmental stages, was determined by quantitative real-time polymerase chain reaction; the transcript levels of AChE were higher in the larvae stage than in other stages. The changes in AChE expression between different developmental stages may be related to their growth habits and metabolism characteristics. This study may offer new insights into the problems of insecticide resistance and insecticide toxicity of nontarget species.