The outer membrane proteins (OMPs) of the marine aquatic animal pathogen Vibrio alginolyticus play an important role in the virulence of the bacterium and are potential candidates for vaccine development. In this study, the major 35.6 kDa OMP of V. alginolyticus was isolated by gel excision from the crude OMP fraction from V. alginolyticus. The sequence of the first 27 amino acid residues from the N-terminal end of the protein is ATV YKD GGT ELL VGG RVE FRG DFI GSD, which has high homology with OmpU proteins from other Vibrio spp. (92%). Lutjanus erythropterus were vaccinated with OmpU, and immunogenicity was confirmed by subsequent western blotting. Enzyme-linked immunosorbent assay (ELISA) analysis demonstrated that OmpU produced an observable antibody response in all sera of the vaccinated fish. L. erythropterus vaccinated with OmpU produced specific antibodies, and were highly resistant to infection with virulent V. alginolyticus. These results indicate that OmpU is an effective vaccine candidate against V. alginolyticus for L. erythropterus.