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BACKGROUND The diamondback moth, Plutella xylostella is a notorious pest of brassicaceae crops globally and has developed serious resistance to insecticide. Insects primarily rely on their innate immunity to defense against various pathogens. In this study, we investigated the immunological functions of a β-1, 3-glucan binding protein from P. xylostella (PxβGBP) and evaluated its potential for biocontrolling P. xylostella. RESULTS The open reading frame of PxβGBP is 1422 bp encoding 473 amino acids residues. PxβGBP contained a CBM39 domain, a PAC domain and a GH16 domain and shared evolutionary conservation with other lepidoptera βGRPs. PxβGBP was strongly expressed in the third instar larvae and fat body. PxβGBP transcript levels increased significantly after the challenge with microbes, including Isaria cicadae, Escherichia coli and Staphylococcus aureus. PxβGBP was identified in P. xylostella larvae challenged by I. cicadae, but not in the naïve insects. Recombinant PxβGBP can directly bind fungal and bacterial cells, and also agglutinate the cells of I. cicadae, S. aureus and E. coli in a zinc-dependent manner. Knockdown of PxβGBP via RNA interference significantly down-regulated the expression of antimicrobial peptide gene gloverin, and enhanced the susceptibility of P. xylostella to I. cicadae infection, leading to high mortality. CONCLUSION These results indicated that PxβGBP plays an important role in the immune response of P. xylostella against I. cicadae infection, and could serve as a potential novel target for pest control. This article is protected by copyright. All rights reserved.