Cyt2Aa2 is a cytolytic and mosquito larvicidal toxin produced by Bacillus thuringiensis subsp. darmstadiensis. The toxin contains 3 tryptophan residues at positions 132, 154, and 157. To study the role of tryptophan on protein structure and functions, each tryptophan residue was substituted by phenylalanine and other different amino acids. Expression test in Escherichia coli showed that all mutant proteins were highly produced as inclusion bodies similar to that of the wild type. The mutant W157F showed haemolytic and mosquito larvicidal activities comparable to the wild type but the mutant W157V and all other mutants at positions 132 and 154 have completely lost these activities. Solubilization and proteinase K activation tests indicated that aromatic residue is required at position 157 and tryptophan residues at positions 132 and 154 are critical residues playing important role to maintain structure and functions of the protein and cannot be changed to any other amino acid.