Cyt2Aa2 is a mosquito-larvicidal protein produced as a 29 kDa crystalline protoxin from Bacillus thuringiensis subsp. darmstadiensis. To become an active toxin, proteolytic processing is required to remove amino acids from its N- and C-termini. This study aims to investigate the functional role of amino acid residues on the N-terminal ${eta}1$ and C-terminal ${alpha}F$ of Cyt2Aa2 protoxin. Mutant protoxins were constructed, characterized and compared to the wild type Cyt2Aa2. Protein expression data and SDS-PAGE analysis revealed that substitution at leucine-33 (L33) of ${eta}1$ has a critical effect on dimer formation and structural stability against proteases. In addition, amino acids N230 and I233-F237 around the C-terminus ${alpha}F$ demonstrated a crucial role in protecting the protoxin from proteolytic digestion. These results suggested that ${eta}1$ and ${alpha}F$ on the Nand C-terminal ends of Cyt2Aa2 protoxin play an important role in the molecular interaction and in maintaining the structural stability of the protoxin.