Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus has emerged as a severe bacterial disease of cultured shrimp. To identify the key virulence factors, two AHPND-causing V. parahaemolyticus (VpAHPND ) strains (123 and 137) and two non-VpAHPND strains (HZ56 and ATCC 17082) were selected. Challenge tests showed that the four strains exhibited different virulence towards shrimp with cumulative mortalities at 48 h postinfection (hpi) ranging from 10 to 92%. The expression of pirABVP in strain 123 and 137 was not significantly different. Genomic analysis revealed that the two VpAHPND strains contain a plasmid with the PirABVP toxins (pirABVP ) flanked by the insertion sequence (ISVal1) that has been identified in various locations of chromosomes in VpAHPND strains. The two VpAHPND strains possessed almost identical virulence factors, while ISVal1 disrupted three genes related to flagellar motility in strain 137. Phenotype assay showed that strain 123 possessed the highest growth rate and swimming motility, followed by strain 137, suggesting that the disruption of essential genes mediated by ISVal1 significantly affected the virulence level. Transcriptome analysis of two VpAHPND strains (123 and 137) further suggested that virulence genes related to the capsule, flagella and primary metabolism were highly expressed in strain 123. Here for the first time, it is demonstrated that the virulence of VpAHPND is not only determined by the expression of pirABVP , but also is mediated by ISVal1 which affects the genes involved in flagellar motility and primary metabolism. The genomic and transcriptomic analysis of VpAHPND strains provides valuable information on the virulence factors affecting the pathogenicity of VpAHPND. © 2020 The Society for Applied Microbiology.