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Abstract Background Multifocal spherical nonstaining cavities and gram-positive, rod-shaped, and endospore-forming bacteria were found in the liver of a sow that died suddenly. Clostridium novyi type B was identified and isolated from the sudden death case, and the isolate was characterized by molecular analyses and bioassays in the current study. Results C. novyi was isolated from the liver of a sow that died suddenly and was confirmed as C. novyi type B by differential PCR. The C. novyi isolate fermented glucose and maltose and demonstrated lecithinase activity, and the cell-free culture supernatant of the C. novyi isolate exhibited cytotoxicity toward Vero cells, demonstrating that the isolate produces toxins. In addition, whole-genome sequencing of the C. novyi isolate was performed, and the complete sequences of the chromosome (2.29 Mbp) and two plasmids (134 and 68 kbp) were identified for the first time. Based on genome annotation, 7 genes were identified as glycosyltransferases, which are known as alpha toxins; 23 genes were found to be related to sporulation; 12 genes were found to be related to germination; and 20 genes were found to be related to chemotaxis. Conclusion C. novyi type B was isolated from a sow in a sudden death case and confirmed by biochemical and molecular characterization. Various virulence-associated genes were identified for the first time based on whole-genome sequencing.