Bacillus thuringiensis Cry3Aa toxin is a coleopteran specific toxin highly active against Colorado Potato Beetle (CPB).We have recently shown thatCry3Aa toxin is proteolytically cleaved by CPBmidgut membrane associated metalloproteases and that this cleavage is inhibited by ADAMmetalloprotease inhibitors. In the present study, we investigated whether the Cry3Aa toxin is a calmodulin (CaM) binding protein, as it is the case of several different ADAMshedding substrates. In pull-down assays using agarose beads conjugated with CaM, we demonstrated that Cry3Aa toxin specifically binds to CaMin a calcium-independent manner. Furthermore, we used gel shift assays and 1H NMRspectra to demonstrate that CaMbinds to a 16-amino acid synthetic peptide corresponding to residues N256-V271 within the domain I of Cry3Aa toxin. Finally, to investigate whether CaM has any effect on Cry3Aa toxin CPBmidgut membrane associated proteolysis, cleavage assays were performed in the presence of the CaM-specific inhibitor trifluoperazine. We showed that trifluoperazine significantly increased Cry3Aa toxin proteolysis and also decreased Cry3Aa larval toxicity.