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Acute hepatopancreatic necrosis disease (AHPND) in shrimp is caused by <i>Vibrio</i> strains that harbor a pVA1-like plasmid containing the <i>pirA</i> and <i>pirB</i> genes. It is also known that the production of the PirA and PirB proteins, which are the key factors that drive the observed symptoms of AHPND, can be influenced by environmental conditions and that this leads to changes in the virulence of the bacteria. However, to our knowledge, the mechanisms involved in regulating the expression of the <i>pirA</i>/<i>pirB</i> genes have not previously been investigated. In this study, we show that in the AHPND-causing <i>Vibrio parahaemolyticus</i> 3HP strain, the <i>pirA^vp</i> and <i>pirB^vp</i> genes are highly expressed in the early log phase of the growth curve. Subsequently, the expression of the PirA<i>^vp</i> and PirB<i>^vp</i> proteins continues throughout the log phase. When we compared mutant strains with a deletion or substitution in two of the quorum sensing (QS) master regulators, <i>luxO</i> and/or <i>opaR</i> (<i>luxO^D47E</i>, Δ<i>opaR</i>, Δ<i>luxO,</i> and Δ<i>opaR</i>Δ<i>luxO</i>), our results suggested that expression of the <i>pirA^vp</i> and <i>pirB^vp</i> genes was related to the QS system, with <i>luxO</i> acting as a negative regulator of <i>pirA^vp</i> and <i>pirB^vp</i> without any mediation by <i>opaR^vp</i>. In the promoter region of the <i>pirA^vp</i>/<i>pirB^vp</i> operon, we also identified a putative consensus binding site for the QS transcriptional regulator AphB. Real-time PCR further showed that <i>aphB^vp</i> was negatively controlled by LuxO<i>^vp</i>, and that its expression paralleled the expression patterns of <i>pirA^vp</i> and <i>pirB^vp</i>. An electrophoretic mobility shift assay (EMSA) showed that AphB<i>^vp</i> could bind to this predicted region, even though another QS transcriptional regulator, AphA<i>^vp</i>, could not. Taken together, these findings suggest that the QS system may regulate <i>pirA^vp/pirB^vp</i> expression through AphB<i>^vp</i>.