Aeromonas veronii is a common human and animal co-pathogenic bacterium that causes serious damage to the aquaculture industry every year. In recent years, the structure of bacterial pili has been found to be an important pathogenic factor. The mshK gene belongs to mannose-sensitive hemagglutinin type IV pili, a structure essential for bacterial adhesion and motility. To investigate the function of mshK in A. veronii, we constructed mshK gene deletion strain (ΔmshK-AV) and complement strain (CmshK-AV) using the effective suicide plasmid-mediated homologous recombination method. Pathogenicity test showed that the median lethal dose value of ΔmshK-AV in Carassius auratus was 2.31 times higher than that of wild-type A. veronii (WT-AV). Compared to WT-AV, ΔmshK-AV showed no significant changes in growth, hemolytic and swarming activities, while biofilm formation capacity was significantly reduced and swimming ability was significantly enhanced. The biological characteristics of CmshK-AV were greatly restored compared to ΔmshK-AV, suggesting that the changes in these characteristics were caused by the deletion of the mshK gene. Quantitative real-time PCR results suggested that the expression of most of the genes related to type IV pili and outer membrane proteins were significantly down-regulated in ΔmshK-AV. Transmission electron microscope results showed that the pili of ΔmshK-AV were sparser than those of WT-AV and CmshK-AV. In conclusion, mutation in the mshK gene may lead to reduced pathogenicity by decreasing biofilm-forming capacity, pili synthesis, and expression of virulence-related genes.