Proteinaceous insecticidal proteins, Cry proteins, from Bacillus thuringiensis (Bt) are insecticidal proteins that are highly active against several species of Lepidoptera. Thus, cry genes encoding these Cry proteins have been widely applied for construction of transgenic crops resistant to pest insects. In this study, through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of Spodoptera exigua and Ostrinia furnacalis were identified. We used site-directed mutagenesis to improve the insecticidal activity of Mod-Cry1Ac, resulted 31 mutant cry genes. These mutant cry genes encodes potent insecticidal proteins in the form of crystalline protoxins of 95 kDa. SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded into polyhedra and activated stably to 65 kDa by trypsin. When the insecticidal activities of these mutant Cry proteins against to larvae of P. xylostella, S. exigua and O. furnacalis were assayed, they showed higher or similar insecticidal activity compared to those of Cry1Ac and Cry1C. Especially, Mutant-N16 is considered to have the potential for the efficacious biological insecticide since it showed the highest insecticidal activity.