Bacillus thuringiensis (Bt) (Berliner) strain LLP29 produces a crystal protein Cyt1Aa6 toxic to mosquito vectors of human diseases. However, the susceptibility of Culex quinquefasciatus (Say) in the current study was 8.25 times higher than that of Aedes albopictus (Skuse) with this single protein Cyt1Aa6 purified from LLP29. To understand the mechanism of the novel mosquitocidal protein, the binding characteristic of brush border membrane vesicles from the two tested mosquitoes was investigated. Enzyme-linked immunosorbent assay showed that Cyt1Aa6 bound to the two mosquitoes’ brush border membrane vesicles. However, the titer of Ae. albopictus was a little higher than that of Cx. quinquefasciatus, with 3.21 and 2.91, respectively. Ligand Western blot analysis showed Cyt1Aa6 toxin specifically bound to the same three proteins (i.e., 68, 54, and 26 kDa) in the two mosquitoes, but one another protein, approximately to 37 kDa, could just be detected in Cx. quinquefasciatus. However, little difference was found in the test of immunohistochemistry. Cyt1Aa6 was detected in the midguts of both mosquitoes with histopathological changes. It would of great importance to the knowledge of the novel toxin against to Cx. quinquefasciatus and Ae. albopictus.