Vibrio splendidusis an opportunistic pathogen that causes skin ulcer syndrome and results in huge losses to theApostichopus japonicusbreeding industry. Ferric uptake regulator (Fur) is a global transcription factor that affects varieties of virulence-related functions in pathogenic bacteria. However, the role of theV. splendidus fur(Vsfur) gene in the pathogenesis ofV. splendidusremains unclear. Hence, we constructed aVsfurknock-down mutant of theV. splendidusstrain (MTVs) to investigate the role of the gene in the effect of biofilm, swarming motility, and virulence onA. japonicus. The result showed that the growth curves of the wild-typeV. splendidusstrain (WTVs) and MTVs were almost consistent. Compared with WTVs, the significant increases in the transcription of the virulence-related geneVshppdmRNA were 3.54- and 7.33-fold in MTVs at the OD600of 1.0 and 1.5, respectively. Similarly, compared with WTVs, the significant increases in the transcription ofVsmmRNA were 2.10- and 15.92-fold in MTVs at the OD600of 1.0 and 1.5, respectively. On the contrary, the mRNA level of the flagellum assembly geneVsflicwas downregulated 0.56-fold in MTVs at the OD600of 1.0 compared with the WTVs. MTVs caused delayed disease onset time and reducedA. japonicusmortality. The median lethal doses of WTVs and MTVs were 9.116 × 106and 1.658 × 1011CFU·ml−1, respectively. Compared with WTVs, the colonization abilities of MTVs to the muscle, intestine, tentacle, and coelomic fluid ofA. japonicuswere significantly reduced. Correspondingly, the swarming motility and biofilm formation in normal and iron-replete conditions were remarkably decreased compared with those of WTVs. Overall, these results demonstrate that Vsfur contributes to the pathogenesis ofV. splendidusby regulating virulence-related gene expression and affecting its swarming and biofilm formation abilities.