The Cry4A toxin is a dipteran-specific insecticidal protein produced by Bacillus thuringiensis subsp. israelensis as a protoxin of 130 kDa. Its active form is a heterodimer of 20- and 45-kDa fragments which is generated by an interhelical cleavage of a 60-kDa intermediate at the position of Gln236 between α5 and α6 helices in domain I. On the other hand, Cry1Aa, which is also produced as a 130-kDa protoxin but toxic to lepidopteran larvae, was processed into the active 60-kDa fragment with no additional cleavage. To investigate the role of the intramolecular cleavage of Cry4A for its insecticidal activity, the loop between α5 and α6 of Cry4A which includes the cleavage site was substituted for the corresponding region of Cry1Aa. The resulting mutant designated GST-60Loop was expressed as a GST-fusion protein. A difference of the processing profile was observed between GST-60 and GST-60Loop in the in vitro digestion assay by trypsin, and the insecticidal activity of GST-60Loop was two-fold lower than that of GST-60. These results suggested that the interhelical cleavage of Cry4A promoted the toxicity against C. pipiens larvae.