One nicotinic acetylcholine receptor non-alpha subunit was cloned from the pond wolf spider, Pardosa pseudoannulata, an important predatory enemy of some insect pests with agricultural importance, such as the green peach aphid Myzus persicae. The subunit shows high amino acid identities to insect beta1 subunits (74-78%), and was denoted as Ppbeta1. Although high identities are found between Ppbeta1 and insect beta1 subunits, amino acid differences are found within loops D, E and F, important segments contributing to ligand binding. The effects of amino acid differences within these loops were evaluated by introducing loops of insect or spider beta1 subunits into rat beta2 subunit and co-expressing with insect alpha subunit. The corresponding regions of rat beta2 chimera beta2(Mpbeta1) (beta2 with loops D, E and F from M. persicae beta1 subunit Mpbeta1) were replaced by loops D, E and F of Ppbeta1 singly or together to construct different chimeras. When these chimeras were co-expressed with insect Nlalpha1, it was found that the replacement of loops D, E and F of beta2(Mpbeta1) by that of Ppbeta1 resulted in a right-ward shift of the imidacloprid dose-response curves, reflecting increases in EC(50), compared to Nlalpha1/beta2(Mpbeta1). By contrast, the influences on ACh potency were minimal. The further study showed that R81Q, N137G and F190W differences, within loops D, E and F respectively, contributed mainly to these sensitivity changes. This study contributes to our understanding of the molecular mechanism underlying selectivity of neonicotinoids against insects over spiders.