Bacillus thuringiensis(Bt) and transgenic crops carryingcrygenes arewidely used in the management of lepidopteran and coleopteranpests. However, almost none of the Cry toxins have insecticidalproperties against sap-sucking insects, such as planthoppers,leafhoppers and aphids. To understand the low insecticidalactivity of Cry1Ac toxin on sap-sucking insects, we investigatedtwo critical steps in the Bt-intoxication cascade: the proteolyticprocessing of Cry1Ac toxin by gut proteases, and the binding ofCry1Ac to brush border membrane vesicles (BBMV) ofNilaparvatalugens. Proteolytic processing of Cry1Ac protoxin byN. lugensgutproteases resulted in an∼65 kDa product, similar to the expectedsize of the trypsin-activated Cry1Ac toxin. In addition, activation ofcysteine proteases inN. lugensgut increased the efficiency ofproteolytic activities in the processing of Cry1Ac. However,feedingN. lugensnymphs with either Cry1Ac protoxin or trypsin-activated Cry1Ac toxin resulted in low mortalities. The LC50ofCry1Ac protoxin and trypsin-activated Cry1Ac was 198.92 and450.18μg/mL, respectively.In vitrobinding analysis of BBMV withthe pre-activated Cry1Ac showed that Cry1Ac toxin couldspecifically bind to the BBMV. However, binding competition with500-fold molar excess GalNAc (N-acetyl-D-galactosamine)suggested that the binding was not mediated by GalNAc-likeglycoproteins. These results indicate that Cry1Ac toxin could besuccessfully processed by the treatment ofN. lugensgutproteases. However, the binding of Cry1Ac toxin to the midgutbrush border membrane was not mediated by GalNAc-likeglycoprotein. This may be responsible for the low susceptibility ofN. lugensto Cry1Ac