Objective To investigate the biochemical characterization of the carboxylesterase LmCesA1 from Locusta migratoria. Results We expressed recombinant LmCesA1 in Sf9 cells by using the Bac-to-bac baculovirus expression system. Enzyme kinetic assays showed that the K-m values of LmCesA1 for alpha-naphthyl acetate (alpha-NA) and beta-naphthyl acetate (beta-NA) were 0.08 +/- 0.01 mM and 0.22 +/- 0.03 mM, respectively, suggesting that LmCesA1 has a higher affinity for alpha-NA. LmCesA1 retained its enzymatic activity during incubations at pH 7-10 and at 10-30 degrees C. In an inhibition experiment, two organophosphate pesticides (malaoxon and malathion) and one pyrethroid pesticide (deltamethrin) showed different inhibition profiles against purified LmCesA1. Recombinant LmCesA1 activity was significantly inhibited by malaoxon in vitro. UPLC analysis showed that no metabolites were detected. Conclusions These results suggest that overexpression of LmCesA1 enhances malathion sequestration to confer malathion tolerance in L. migratoria.