Abstract: 【Objective】 This study aimed to investigate the biological characteristics of two pathogenic microsporidian isolates of silkworm and reveal the molecular basis for their pathogenicity differences through infection transcriptome analysis， providing theoretical reference for the pebrine disease control and study of pathogenic microsporidian pathoge-nesis of silkworm infection. 【Method】 Spores were enriched from infected silkworm excrement and eggs using the saturated salt solution method. Biological characteristics were identified by morphological observation， median lethal dose （LD₅₀） determination， small subunit ribosomal DNA （SSU rDNA） sequence analysis， and phylogenetic tree construction. Differen-tially expressed genes （DEGs） of the two microsporidia isolates after infecting silkworms were compared by transcriptome sequencing and bioinformatics analysis， and real-time fluorescence quantitative PCR was used for validation. 【Result】 The saturated salt solution method could efficiently enrich spores with few background impurities， making it suitable for early population monitoring. GXND-15 spores exhibited diverse morphology， including oval， elliptical， and rod-shaped heterogeneous forms， with the LD₅₀ of 1×10^-5.19； GXNP-24 spores had relatively uniform and regular morphology， and theywere predominantly elliptical， with the LD₅₀ of 1×10^-6.27. The SSU rDNA sequences of two microsporidian isolates showed a similarity of 99.6%， all belonged to Nosema. Screening of DEGs revealed 2831 and 1372 DEGs in the GXND-15 and GXNP-24 infection groups respectively， and the DEGs were annotated to GO functional terms of catalytic activity， binding， cellular process， metabolic process， biological regulation， and cellular anatomical entity； DEGs in the GXND-15 infection group were primarily enriched in signaling pathways including ribosome， ECM-receptor interaction， insect hormone biosynthesis， oxytocin signaling pathway， as well as starch and sucrose metabolism； DEGs in the GXNP-24 infection group were primarily enriched in signaling pathways including insect hormone biosynthesis， ascorbate acid and aldarate metabolism， pentose and glucuronate interconversions， retinol metabolism， and steroid biosynthesis. Real-time fluorescence quantitative PCR validation showed that the trends of relative expression of five DEGs were consistent with the transcriptome sequencing analysis results. 【Conclusion】 Saturated salt flotation method can efficiently enrich microsporidian spores from silkworm excrement and eggs and improve microscopic sensitivity， making it suitable for early population monitoring in production. GXND-15 and GXNP-24 belong to Nosema and are closely related， but exhibit marked differences in spore morphology， virulence， and host transcriptional response profiles. Endemic area， host species， and generation history are important factors driving the differentiation of microsporidian biological characteristics and directly determine their embryogenic infectivity.