Abstract: The Chinese equine infectious anemia virus (EIAV) donkey-leukocyte attenuated vaccine (DLV) provides a unique natural model system, by which the mechanism of virulent attenuation and the immunological control of lentivirus replication can be studied. In this study, we compared gp90 sequences of the env gene from both virulent and attenuated EIAV strains and found that all theses sequences lost the potential N-linked glycosylation sites in the principal neutralization domain of the V3 region of the attenuated envelope gp90. To determine whether the loss of this potential N-linked glycosylation site also changes the neutralization sensitivity of the vaccine strain, this glycosylation site was restored in the infectious clone pLGFDg5. The derived virus, pLGFDg5V, demonstrated a decreased replication rate in vitro when compared with the parental vaccine strain. More importantly, the reverse mutation of the glycosylation site in the V3 region significantly enhanced the resistance of pLGFDg5V to serum neutralizing antibodies, which can recognize parental viruses. This study improves the understanding of the mechanism that the Chinese EIAV vaccine provides solid immunity to the infection of virulent strains.