Objective To better comprehend the molecular structure and physiological function of the housefly larval peritrophic matrix(PM), a mass spectrometry approach was used to investigate the PM protein composition. Methods The PM was dissected from the midgut of the third instar larvae, and protein extracted from the PM was evaluated using SDS-PAGE. A 1D-PAGE lane containing all protein bands was cut from top to bottom, the proteins in-gel trypsinised and analysed via shotgun liquid chromatography- tandem mass spectrometry(LC-MS/MS). Results In total, 374 proteins, with molecular weights varying from 8.225 k D to 996.065 k D and isoelectric points ranging from 3.83 to 11.24 were successfully identified, most identified proteins were mainly related to immunity, digestion, nutrient metabolism and PM structure. Furthermore, many of these proteins were functionally associated with pattern binding, polysaccharide binding, structural constituent of peritrophic membrane and chitin binding, according to Gene Ontology annotation. Conclusion The PM protein composition, which provides a basis for further functional investigations of the identified proteins, will be useful for understanding the housefly larval gut immune system and may help to identify potential targets and exploit new bioinsecticides. Objective To better comprehend the molecular structure and physiological function of the housefly larval peritrophic matrix (PM), a mass spectrometry approach was used to investigate the PM protein composition. Methods The PM was dissected from the midgut of the third instar larvae, and the protein extraction from the PM was evaluated using SDS-PAGE. A 1D-PAGE lane containing all protein bands was cut from top to bottom, the proteins in-gel trypsinised and analysed via shotgun liquid chromatography-tandem mass spectrometry (LC-MS / MS). Results In total, 374 proteins, with molecular weights varying from 8.225 kD to 996.065 kD and isoelectric points ranging from 3.83 to 11.24 were successfully identified, most identified proteins were mainly related to immunity, digestion, nutrient metabolism and PM structure. Further, many of these proteins were functionally associated with pattern binding, polysaccharide binding, structural constituent of peritrophic membrane and chitin binding, accordi ng to Gene Ontology annotation. Conclusion The PM protein composition, which provides a basis for further functional investigations of the identified proteins, will be useful for understanding the housefly larval gut immune system and may help identify potential targets and exploit new bioinsecticides.