Bacillus thuringiensis (Bt) is the most widely used biological insecticide for the control of forest and agricultural pests and vectors of human and animal disease. Bt is a ubiquitous spore-forming bacterium that is capable of killing insect larvae largely through the action of a protein known as crystal δ-endotoxin. The mechanism of killing has generally been thought to be through toxin-mediated lysis of the gut epithelial cells, which eventually leads to starvation or septicemia due to B. thuringiensis growth in the hemocoel. Here we report that B. thuringiensis does not induce septicemia itself, but depends on enteric bacteria to kill gypsy moth larvae. Elimination of the microbial community by antibiotic feeding abolished Bt insecticidal activity, and re-establishment of an Enterobacter sp., an indigenous enteric of gypsy moth, restored Bt killing ability. Experiments using an E. coli expressing Bt toxin validate that the loss of killing by Bt in insects fed antibiotics was not due to the direct action of antibiotics on Bt, consistent with our proposed model. Enterobacter sp. was capable of growing to high populations in hemolymph withdrawn from gypsy moth larvae whereas Bt was not. Additional data indicate that gut bacteria also contribute to Bt-associated killing in other lepidopteran species, including Manduca sexta, Vanessa cardui, Pieris rapae, and Heliothis virescens. The role of enteric bacteria in Bt-induced killing and the general applicability of this model are discussed.