Abstract Grapholita molesta is one of the most damaging pests worldwide in stone and pome fruits. Application of chemical pesticides is still the main method to control this pest, which results in resistance to several types of insecticides. Carboxylesterase (CarE) is one of the important enzymes involved in the detoxification metabolism and tolerance of xenobiotics and insecticides. However, the roles of CarEs in insecticides susceptibility of G. molesta are still unclear. In the present study, the enzyme activity of CarEs and the mRNA expression of six CarE genes were consistently elevated after treatment with three insecticides (emamectin benzoate, lambda‐cyhalothrin, and chlorantraniliprole). According to spatio‐temporal expression profiles, six CarE genes expressed differently in different developmental stages, and highly expressed in some detoxification metabolic organs. RNAi‐mediated knockdown of these six CarE genes indicated that the susceptibility of G. molesta to all these three insecticides were obviously raised after GmCarE9 , GmCarE14 , GmCarE16 , and GmCarE22 knockdown, respectively. Overall, these results demonstrated that GmCarE9 , GmCarE14 , GmCarE16 , and GmCarE22 play a role in the susceptibility of G. molesta to emamectin benzoate, lambda‐cyhalothrin, and chlorantraniliprole treatment. This study expands our understanding of CarEs in insects, that the same CarE gene could participate in the susceptibility to different insecticides.