[Objective] The objectives are to identify a parasitic Hirsutella Pat. of Bombyx mori Linnaeus. and to research the spore culture of this fungus. [Methods] The comparison of morphological characteristics and internal transcribed spacer (ITS) sequence analysis were used for identification of specimens. single factor test and orthogonal test were carried out for studying sporulation conditions optimization. [Results] According to the morphological characterization and phylogenetic tree, the fungus was identified as Hirsutella satumaensis Aoki. The optimum conditions for sporulation is: peptone 3%, glucose 1%, silkworm chrysalis powder 1.5%, vitamin B1 1%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.1%, agar 2%, distilled water 1 000 ml, 25 °C. [Conclusion] H. satumaensis Aoki, a known species in China, which microscopic characteristics were described again and the associated molecular systematics data were added in this paper. The sporulation conditions optimization could provide a reference for the spores acquisition and application of this group of fungi.