Aims: To investigate the virulence of the Vp_PirAB-like genes in Vibrio parahaemolyticus-acute hepatopancreatic necrosis disease (AHPND)-causing strain and the factors that are associated with the virulence level. Methods and Results: The virulence of Vp_PirAB-like was examined using a non-virulent strain FP11 of V. parahaemolyticus transformed with a plasmid harbouring Vp_PirAB-like genes and then it was used to challenge shrimp Litopenaeus vannamei and Marsupenaeus japonicus. Both species experienced 100% mortality at 10 days post infection. Analysis of a mutant strain (E1M), that was originally identified as virulent strain (E1) but lost its virulence to L. vannamei, revealed that it lacked a part of the Vp_PirA-like gene and all of the Vp_PirB-like gene. The copy numbers of Vp_PirA-like and Vp_PirB-like genes varied among virulent strains and were not correlated with their virulence. In Western blotting, Vp_PirA-like and Vp_PirB-like proteins were detected in both the cell lysate and the culture supernatant. The strongest intensity of detecting band in the culture supernatant was observed in the strain that caused the highest mortality. The V. parahaemolyticus AHPND-causing strain, unlike the human tdh-positive strain, did not show any enterotoxicity. Conclusion: Vibrio parahaemolyticus AHPND-causing strains secrete the Vp_PirA-like and Vp_PirB-like proteins during the growing phase. The amount of secreted proteins affects the shrimp mortality. Significance and Impact of the Study: The secreted proteins of Vp_PirAB-like are key factors of virulence in the V. parahaemolyticus AHPND-causing strain, but not gene copy.