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The pathogenic <i>Bacillus cereus</i> strain XS0724 isolated from China can cause high lethality to <i>Trionyx sinensis</i>, but little information is available on their detailed interactions. In this study, histopathologic profiling indicated that <i>B. cereus</i> caused vacuolization and cell necrosis in the liver, spleen, kidney, and intestine. The identification of the virulence factor genes non-hemolytic enterotoxin (<i>Nhe</i>), hemolysin BL (<i>Hbl</i>), and enterotoxin FM (<i>entFM</i>) confirmed bacterial pathogenicity. Splenic transcriptomic sequencing at 96 h post-infection identified various immune-related genes mapped to diverse gene families, including interleukin, complement, chemokine, and interferon. The differentially expressed genes (DEGs) were enriched in 2174 GO terms: 1694 in biological processes, 138 in cellular components, and 342 in molecular functions. Further KEGG enrichment indicated that DEGs were primarily associated with the phagosome, NF-kappa B signaling pathway, and PI3K-Akt signaling pathway. The DEGs and enriched pathways may be involved in the elimination of invasive <i>B. cereus</i>. These data laid the foundation for elucidating the potential molecular mechanisms in this bacterial infection process, and provided robust genetic evidence for subsequent work on resistance genes of <i>T. sinensis</i>.