Sugarcane giant borer (Telchin licus licus) is a serious sugarcane pest in Americas whose endophytic lifestyle hampers effective chemical and biological controls. Therefore, development of alternative control methods is extremely important. Envisaging development of transgenic plants resistant to this pest, we investigated the effect of the Bacillus thuringiensis Cry protein Cry1Ia12synth (truncated protein lacking C-terminus with plant codon usage) and variants against T. l. licus. cry1Ia12synth gene was used to generate mutated variants, which were screened for toxicity toward T. l. licus. For that purpose, an innovative technique combining cry gene shuffling with phage-display was used to build a combinatorial library comprising 1.97x10(5) Cry1Ia12synth variants. Screening of this library for variants binding to T. l. licus Brush Border Midgut Vesicles led to the identification of hundreds of clones, out of which 30 were randomly chosen for toxicity testing. Bioassays revealed four variants exhibiting activity against T. l. licus as compared to the non-toxic Cry1Ia12synth. Eight single substitutions sites were found in these active variants. Based on theoretical molecular modelling, the probable implications of these mutations are discussed. Therefore, we have four genes encoding Cry1Ia12synth variants active against T. l. licus promising for future development of resistant transgenic sugarcane lines.