An isolated virulence Riemerella anatipestifer strain passaged 200 times on TSB agar were used for the virulent to avirulent conversion. The effects of passage on biological properties of outer membrane proteins (OMPs) were investigated using the virulent and avirulent strains. Transmission electron microscopy demonstrated that the avirulent strain produced lower amounts of outer membrane vesicles and the outer membrane decreased, the cytoplasmic appearance jumbled. The OMPs of the virulent strain agglutinated only in RA serotype 2 antisera, whereas the OMPs of the avirulent strain agglutinated in antisera of RA 1, 2, 10 and 11. SDS-PAGE Analysis showed the OMPs profiles of both strains were similar but the immunoblotting profiles were different. The protective immunity against Riemerella anatipestifer infection was investigated by immunizations with OMPs in ducks. ELISA results showed that the OMPs induced the production of antibodies in immunized ducks, but the OMPs of virulence strain induced higher antibody titers than the attenuated strain (P < 0.05). RA2 group showed significantly higher survival rates (100%) than RA200 group (0%) after challenged with the homologous virulent strain. The ompA gene of both stains were also amplified by PCR, nucleotide homology was 99.9%. In conclusion, OMPs of virulent RA strain are suitable candidates for vaccine development. Biological properties of OMPs undergoes significant changes during serial passage and suggest that vigilance should be used when extrapolating data obtained from the study of high-passage strains.