The microsporidian, Nosema antheraeae can infect Chinese oak silkworm, Antheraea pernyi, and cause pebrine disease. This disease could greatly reduced the income from tussah industry. For seeking a way to overcome this disease, more and more researchers were worked for exploring the infection mechanism of the spores. Here, in order to identify the metabolites of N. antheraeae for analyzing key metabolic pathways, Gas chromatography-mass spectrometry (GC/MS)-based metabolome was performed. 1975 metabolic characteristic peaks were assignment and finally fifty-two metabolites were identified, including amino acid, fatty acid, sugars, etc. Database searches showed that the monoisotopic mass of the obtained metabolites ranged from 57 to 539 Da. Metabolic pathway enrichment analysis revealed that many basic metabolism processes, including protein biosynthesis, amino acid metabolism, pentose phosphate pathway, etc were found. These results, for the first time, provided a tool to explore the metabolic profiling of the microspridia and may provide a new horizon for seaking pathogenic molecules during infection of spores.