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Exploiting specific targets is of specific interest in developing eco-friendly pesticides. We isolated, purified and characterized a novel β-N-acetyl-D-hexosaminidase (OfHex1) from the fifth instar larva integument of the Asian corn borer, Ostrinia furnacalis (Guenée). OfHex1 was purified 1468-fold to homogeneity with an activity yield of 20% by four column chromatography steps. Under denaturing conditions, the molecular mass of OfHex1 was determined to be 67.0 kDa by MS and SDS/PAGE, but 128 kDa by gel filtration chromatography, suggesting that it was in the form of a homodimer. Its pI was 4.7 as determined by IEF electrophoresis. OfHex1 was shown to be an exo-splitting enzyme, acting by cutting one β-GlcNAc unit once from the nonreducing ends of substrates, with a preference for shorter substrates. OfHex1 could hydrolyze p-nitrophenyl β-GlcNAc, p-nitrophenyl β-GalNAc and chito-oligosaccharides (degree of polymerization from 2 to 6), but it could not hydrolyze the complex N-glycan substrate (GlcNAcβ-1,2Manα-1,6)(GlcNAcβ-1,2Manα-1,3)Manβ-1,4GlcNAcβ-1,4GlcNAc-PA as well as the long polymer chitin. Certain structural elements of substrates, the 2-acetamido group and the β-glycoside bond linkage, were determined to be essential for its activity. The 2.6 kb cDNA encoding OfHex1 was obtained by RT-PCR. Sequence analysis indicated that it was different from reported β-N-acetyl-D-hexosaminidases with N-glycan hydrolytic activities. Real-time PCR determined that its transcriptional level increased dramatically before the molting stage. According to its hydrolytic mode, substrate spectrum, cDNA sequence and mRNA transcriptional level, we deduced that OfHex1 could be mostly involved in insect chitin catabolism and might be a specific target for pesticide development.