The cadherin-like protein Bt-R₁ is a Bt Cry1A-binding protein distributed on the midgut epithelia of tobacco hornworm (Manduca sexta) larvae. The CR12-MPED peptide is a 23.3 kDa truncation of the extracullar portion of Bt-R₁. This peptide acts as a synergist for Bt Cry insecticidal proteins when ingested with Cry toxins by lepidopteran larvae. Currently, we are investigating mechanisms that may explain the effect of toxicity enhancement by CR12-MPED. We report that the CR12-MPED peptide was unfolded in structure as determined by far-UV circular dichroism and ¹H-NMR spectroscopy. Histomicroscopy demonstrated that the CR12-MPED peptide bound to whole midgut microvilli from M. sexta larvae. CR12-MPED binds Cry1A toxins. However, the addition of CR12-MPED peptide did not change the binding localization of Cry1A toxins on insect midgut. We propose that the CR12-MPED peptide accumulated on insect microvilli may attract Cry1A molecules to attack these places via peptide-toxin binding hence the toxicity of Cry1A is potentiated. Truncations of CR12-MPED were constructed and expressed in E. coli, and the CR12 sub-region had the same ability as the original peptide to increase Cry1Ac toxicity against corn earworm (Helicoverpa zea) larvae. The application of CR12-MPED or CR12 peptide will enable Bt toxin to effectively control lepidopteran pests at a lower dosage, thus extending the effective host range of the Cry toxin against pest insects.