Bt toxins kill insects by binding to various receptors at insect midgut and creating pores on the midgut membranes. However, the application of Bt toxins in agriculture is threatened by evolved resistance of insects. Our understanding of molecular basis in insects involved in Bt toxin binding is incomplete, especially in diamondback moth (DBM, Plutella xylostella), a major agricultural pest. Identifying Bt toxin receptors has remained challenging due to lacking sufficiently sensitive methods. Here, we report a novel technique, on-membrane capture, that identified numbers of previously unknown proteins, in addition to known receptors like cadherin and APN2, from the DBM midgut as binding partners of three Bt toxins Cry1Ac, Cry1Ab, and Cry1Bd. Among them, glucosinolate sulfatases GSS1 and GSS2 are receptors specifically interacted with Cry1Bd. Reduction in GSSs expression increased tolerance of DBM to Bt toxins. Expressing DBM GSSs in silkworms dramatically decreased the tolerance of the transgenic silkworms to Cry1Bd. Therefore, the on-membrane capture provides a new solution to identify Bt toxin receptors in insects.