Vibrio vulnificus biotype 2 serovar E (VSE) is the aetiological agent of both eel vibriosis epizootics and sporadic cases of human vibriosis. We selected two paralogs for UDP-glucose 4-epimerase, corresponding to annotations VV3026 and VV2639 in the genome of the biotype 1 strain YJ016. We demonstrate that VV3026-like and VV2639-like were functionally homologous to gne and galE, respectively. Both genes were present in all V. vulnificus strains tested regardless of biotype or serovar. Two VSE mutants, 4999-2 (gne-deficient) and 4999-3 (galE-deficient), were obtained by allelic exchange. The 4999-2 mutant showed deep alterations in the outer membrane (OM) architecture concomitantly to a noticeable increase in sensitivity to cationic peptides and serum together with the loss of virulence for eels and mice. In contrast, the mutant 4999-3 did not present alterations in either its OM or in surface hydrophobicity, giving the same results as the wild-type strain in all virulence assays. The change to avirulent phenotype produced by mutation in gne was correlated to the loss of the O-antigen LPS. Complementation of gne-deficient mutant restored the LPS structure together with the virulent phenotype. We also demonstrate that Gne is able to perform UDP-GalNAc 4-epimerase activity, as well as UDP-Gal-4-epimerase (GalE) activity, which explain the results obtained with mutant 4999-3 (galE-deficient).