The binary toxin produced from Bacillus sphaericus is highly toxic against mosquito larvae. The two major components of the binary toxin are 42-kDa BinA and 51-kDa BinB. BinA is proposed to function as a toxic subunit, whereas BinB is responsible for receptor binding. So far, there is no detailed knowledge of the molecular mechanism of the binary toxin, mainly due to the lack of structural information. In the absence of the structural information, we investigated the role of amino acids at the Nterminal region of BinB in governing the larvicidal activity via amino acid substitutions at residues spanning positions 35-37 and 41-43. Bioassays, receptor and membrane binding analyses demonstrated that residues P35, F41 and Y42 of BinB are crucial for the larvicidal activity, especially during the steps of membrane and receptor binding. In order to identify functional sites of BinA, aromatic cluster in BinA at positions Y213, Y214, Y215, W222 and W226 were substituted by leucine. All mutants were able to insert into lipid monolayers as observed by Langmuir–Blodgett trough and could permeabilize the liposomes in a similar manner as the wild type. However, mosquito-larvicidal activity was abolished for W222L and W226L mutants suggesting that tryptophan residues at both positions play an important role in the toxicity of BinA, possibly involved in the cytopathological process after toxin entry into the cells. In order to gain more insight into the molecular mechanism of the binary toxin, the X-ray crystallographic analysis was performed. Only the crystal of activated BinB could diffract to 1.75 Å resolution. The structure of activated BinB has been determined by single-wavelength anomalous dispersion (SAD) method. The structure of activated BinB is composed of two domains: an N-terminal - trefoil lectin-like domain and an elongated C-terminal domain. The N-terminal domain shares similar features to the sugar binding proteins or lectins. The C-terminal domain resembles the pore-forming domain of the aerolysin-type β-pore forming toxins. Taken together, the crystal structure of the BinB subunit confirms the important role of BinB for receptor binding and possibly facilitating the internalization of the BinA or BinA-BinB complex into the cells to exert its cytotoxicity. However, the functional detail of BinB protein requires further elucidation.