Binary toxin (Bin) produced by Lysinibacillus sphaericus is toxic to Culex and Anopheles mosquito larvae. It has been used world-wide for control of mosquitoes that vector disease. The Bin toxin interacts with the glucosidase receptor, Cpm1, in Culex and its orthologue, Agm3, in Anopheles mosquitoes. However, the exact mechanism of its mode of action is not clearly understood. It is essential to understand mode of action of Bin toxin to circumvent the resistance that develops over generations of exposure. A suitable model cell line will facilitate investigations of the molecular action of Bin toxin. Here we report Bin toxin activity on Ag55 cell line that has been derived from an actual target, Anopheles gambiae larvae. The Bin toxin, both in pro and active forms, kills the Ag55 cells within 24 h. Bin toxin internalizes in Ag55 cells and also induces vacuolation as tracked by Lysotracker dye. The dose response studies showed that 1.5 nM of Bin toxin is sufficient to induce vacuolation and Ag55 cell death. Presence of α -glucosidase gene (Agm3) expression in the Ag55 cells was also confirmed. Thus, Ag55 cells constitute an appropriate model system to decipher the mode of Bin action in mosquito larvae.