Abstract: Objective Utilizations of arabinose, arginine, lysine and ornithine are the major biochemical tests used to differentiate Vibrio fluvialis from its close species. In this study, we preformed a molecular genetic analysis on an arginine dihydrolase-negative V. fluvialis strain Ma-2531. Methods Polymerase chain reaction (PCR) was performed by using multiple primers specific to the arc operon gene cluster, along with the sequence analysis of the amplicon. Meanwhile, we measured the growth curve and pH variation of the cultures of Ma-2531 and EF85003 which was arginine dihydrolase positive. Results Primer pairs arc-F/arc-R, arc-F/arc-rev and arc-ck-up/arc-R gave negative amplifications which are specific to arcBCA sequence. The primer pairs, arc1-up/arc1-dn and arc72695-up/arc75219-dn, annealing to the flanking sequence of arcBCA gave the expected amplicons, respectively. Sequence analysis of the product of arc1-dnRev/arc72695-upRev revealed the existence of the transposase IS4, resulting in the deletion of an 8.6 kb fragment containing the arcBCAD gene cluster and the up-flanking sequence (the carbamoyl aspartate aminotransferase regulatory subunit gene and the catalytic subunit gene). No differences in the growth curve and pH change were observed between Ma-2531 and EF85003. Conclusion The transposition of transposase IS4 caused the negative phenotype of arginine dihydrolase in V. fluvialis Ma-2531, but it might be a random and rare event, resulting diversity of biochemical metabolism in V. fluvialis.