Aeromonas veronii is a Gram-negative pathogenic bacterium that causes various diseases in aquatic animals and humans, posing a serious threat to aquaculture and public health. The type Ⅵ secretion system (T6SS) is a key virulence factor determining the pathogenicity of A. veronii. It is known that the bacterial enhancer-binding protein (bEBP) VasH is responsible for regulating the T6SS function, while whether this regulatory relationship exists in A. veronii remains unknown. [Objective] To elucidate the impacts of VasH on T6SS expression and function in A. veronii, thereby providing a theoretical basis for deciphering the mechanism of T6SS-mediated pathogenicity of A. veronii and for the subsequent prevention and control of A. veronii infections. [Methods] With A. veronii C4 as the wild-type strain, the VasH-deficient mutant strain ΔvasH and the complemented strain ΔvasH/p-vasH were constructed via a homologous recombination strategy. RT-qPCR was employed to measure the relative expression levels of T6SS genes in each strain to clarify the effect of VasH on T6SS gene expression. Growth curve establishment, transmission electron microscopy (TEM) for observing bacterial morphology, and the crystal violet assay for biofilm quantification were performed to determine the influences of VasH on pathogenic characteristics. An in vitro bacterial competition assay and a zebrafish model for determining the median lethal dose (LD₅₀) were employed to assess the comprehensive effects of VasH on bacterial competitiveness and pathogenicity. [Results] The VasH-deficient mutant strain ΔvasH and the complemented strain ΔvasH/p-vasH were successfully constructed. The deletion of VasH resulted in a decrease (P<0.05) in the expression of genes encoding both T6SS structural and effector proteins, indicating that VasH was responsible for regulating T6SS gene expression. The deletion of vasH did not significantly affect the growth or surface morphology/structure of A. veronii. However, it led to a significant increase in the biofilm formation and a significant decrease in in vitro bacterial competitiveness. The zebrafish infection assay showed that the LD₅₀ of ΔvasH was 1.58×10¹¹, which was 2.15 times that (7.34×10¹⁰) of the wild type. Moreover, at an infection concentration of 5×10⁸ CFU/mL, the death rate of zebrafish decreased from 33.3% in the wild-type infection group to 0 following infection with ΔvasH, indicating that deletion of vasH attenuated the virulence of A. veronii. [Conclusion] In A. veronii, VasH positively regulates T6SS gene expression. It may not be involved in regulating the growth state or external morphology of the pathogen but significantly influences the biofilm formation and competitiveness of A. veronii, thereby modulating the overall virulence during infection of the zebrafish host. This study provides essential research tools and lays a preliminary foundation for further exploration of the molecular mechanisms by which VasH mediates pathogenicity through regulating T6SS activity and function in A. veronii.