Abstract <p>CobB, NAD⁺-dependent lysine deacetylase, may be involved in the complex biological processes, directly or indirectly regulating the physiological activities of bacteria through deacetylation. Currently, the role of CobB in fish pathogen <i>Vibrio alginolyticus</i> is very unclear. This study compared the transcriptional differences by RNA-seq and biological characteristics analysis of wild-type <i>V. alginolyticus</i> with its <i>cobB</i> deletion strain Δ<i>cobB</i>. The RNA-seq results showed that Δ<i>cobB</i> strain had 259 differentially expressed genes (DEGs) compared with WT, of which 78 genes were significantly up-regulated, and 181 genes were significantly down-regulated. These DEGs were mainly enriched in pathways such as cell movement, membrane transport, amino acid metabolism, and GO terms such as cell process and membrane. Phenotypic results indicated that deletion of <i>cobB</i> gene changed the morphology of the bacterial cells, and weakened their migratory ability, biofilm formation ability and extracellular protease activity of <i>V. alginolyticus,</i> but had almost no effect on their growth, which can explain effectively the above differences in RNA-seq results. Compared with WT, LD₅₀ value of Δ<i>cobB</i> increased by 60-fold and the drug resistance also increased. Moreover, deletion of <i>cobB</i> gene decreases the virulence of <i>V. alginolyticus</i>. These results confirmed the possible regulatory mechanism of CobB in cells, and indicated that CobB played an important role in the <i>V. alginolyticus</i> metabolism as well.</p>