Abstract: In the present study, the innate immune response of Plutella xylostella against Paecilomyces cicadae challenges was surveyed using a constructed suppression subtractive hybridization library. A total of 825 P. xylostella ESTs were obtained. They were assembled to 412 unigenes (including 107 contigs and 305 unique sequences). By BLASTN, 28.24% of the P. xylostella ESTs had no hit or hitted on unknown gene. 71.76% ESTs were subjected to annotated and categorized by their putative functions, including immunity-related genes, metal ion binding and processing, nucleic acid and protein metabolism and process, cell signaling, cell structure, shape and mobility, energy metabolism, stress and detoxification and other genes. GeneOntology revealed that 39 immunity-related genes potentially involved in innate immunity and were subdivided into 4 categories: recognition molecules (such as peptidoglycan recognition protein, gram negative bacteria-binding protein), proteases and protease inhibitors (such as trypsin-like serine proteinase and serpin), antimicrobial effectors (antimicrobial peptide, lysozyme and prophenoloxidase-activating proteinase, etc) and other genes. qRT-PCR results showed that the diamond-back moth peptidoglycan recognition protein gene, imaginal disk growth factor gene, gloverin gene, lysozyme gene, prophenoloxidase-activating proteinase 3 gene and transferrin gene were up-regulated after immunization with bacteria and fungus. The expression profiles of these genes showed difference after being induced by bacteria and fungus, indicating they play different biological roles in immunity. These results showed that P. xylostella infected by P. cicadae could express serials of special genes and the response of P. xylostella to P. cicadae infection was a complex process. ESTs categorization analyses allowed us to identify 39 considered candidate immune genes, providing new insights into the molecular mechanisms during the innate immunity of P. xylostella against P. cicadae infection.