This study reports the baculovirus expression and biochemical characterization of recombinant acetylcholinesterase from Haematobia irritans (L.) (rHiAChE) and the effect of the previously described G262A mutation on enzyme activity and sensitivity to selected organophosphates. The rHiAChE was confirmed to be an insect AChE2-type enzyme with substrate preference for acetylthiocholine (K_m 31.3 µM) over butyrylthiocholine (K_m 63.4 µM) and inhibition at high substrate concentration. Enzyme activity was strongly inhibited by eserine (2.3 × 10 ^-10 M), BW284c51 (3.4 × 10^-8 M), malaoxon (3.6 × 10^-8 M), and paraoxon (1.8 × 10^-7 M), and was less sensitive to the butyrylcholinesterase inhibitors ethopropazine (1.1 × 10 ^-6M) and iso-OMPA (4.1 × 10 ^-4 M). rHiAChE containing the G262A substitution exhibited decreased substrate affinity for both acetylthiocholine (K_m 40.9 µM) and butyrylthiocholine (K_m 96.3 µM), and exhibited eight-fold decreased sensitivity to paraoxon, and ∼1.5- to 3-fold decreased sensitivity to other inhibitors. The biochemical kinetics are consistent with previously reported bioassay analysis, suggesting that the G262A mutation contributes to, but is not solely responsible for observed phenotypic resistance to diazinon or other organophosphates.