Acetyl-coenzyme A carboxylase (ACC) catalyses the carboxylation of acetyl-coenzyme A (acetyl-CoA) to produce malonyl-CoA during the de novo synthesis of fatty acids. Spirotetramat, an inhibitor of ACC, is widely used to control a range of sucking insects, including the Aphis gossypii. In the present study, Reverse transcription quantitative real-time PCR (RT-qPCR) results demonstrated that ACC was significantly overexpressed in a laboratory-selected spirotetramat-resistant strain compared with the susceptible strain. ACC RNA interference significantly suppressed fecundity and led to cuticle formation deficiencies in resistant adults and nymphs compared with the control. The full-length ACC gene was sequenced from both resistant and susceptible cotton aphids, and a strong association was found between spirotetramat resistance and 14 amino acid substitutions in the biotin carboxylase domain and carboxyl transferase domain of the ACC gene. Furthermore, ACC activity was higher in resistant aphids than in the susceptible strain, and ACC in the resistant aphids exhibited significant insensitivity to spirotetramat and spirotetramat-enol. The results indicate that the overexpressed insensitive (mutated) ACC target played an important role in the high levels of spirotetramat resistance observed here. This association of amino acid substitution with resistance is the first report of a potential target site mechanism affecting spirotetramat in the cotton aphid.