Proteomic differences between Bemisia tabaci biotypes (B and Q) were investigated by two-dimensional gel electrophoresis in conjunction with mass spectroscopic analysis. Among several protein spots specific to biotype B, carboxylesterase 2 (Coe2) was significantly more expressed in biotype B. Phylogenetic analysis demonstrated the close relationship of Coe2 with Myzus persicae esterase E4. Comparison of full-length cDNA sequences of Coe2 revealed no amino acid differences in functionally important conserved regions between biotypes B and Q. The transcription level of the Coe2 gene (coe2) was 5.8-fold higher in biotype B than in biotype Q, but the coe2 copy number was not different between biotypes, suggesting that the overexpression of Coe2 was due to transcriptional up-regulation. Native isoelectric focusing followed by mass spectrophotometric analysis confirmed that the overexpressed pI 5.7 esterase in biotype B was Coe2. In-gel inhibition of Coe2 by three insecticides indicated the interaction of Coe2 with chlorpyrifos-methyl oxon and permethrin, but not with imidacloprid. These findings suggest that overexpression of Coe2 in biotype B can confer chemical defense against pyrethroid and organophosphate insecticides, perhaps by sequestration and hydrolysis, as seen in M. persicae E4. Finally, utility of Coe2 as a potential biotype-specific protein marker is discussed.