Carboxylesterase-mediated insecticide resistance is a major problem affecting the control of both insect agricultural pests and disease vectors like Culex quinquefasciatus. In C. quinquefasciatus many insecticide resistance carboxylesterases (CBEs) have been identified and a very small number have been isolated from larvae and found to have activity with insecticides. The importance of CBEs in insecticide resistance in C. quinquefasciatus is well established, however, there have been no studies into their structure, in fact, of all insects the only CBE structure determined so far is of LcαE7, a catalytic detoxifier from Lucilia cuprina. In this work, we used lysine methylation to obtain the first structure of a ‘sequestration’ CBE, Cqestβ21, one of the most widely expressed insecticide resistance CBEs in C. quinquefasciatus. Cqestβ21 adopts a canonical α/β-hydrolase fold that has a high similarity with LcαE7 and acetylcholinesterase (AChE), the target of organophosphate (OP) and carbamate insecticides. Comparison with other similar esterases reveals a large binding pocket that was shown to bind the OPs, temephos and paraoxon-ethyl, and the carbamate, propoxur, with Kd values in the nM and µM range respectively. This structure is vital for future studies to better understand the mechanism of CBE-mediated insecticide resistance and combat it through targeted-insecticide design.