Equine infectious anemia virus (EIAV) vaccine is the first successfully applied lentiviral vaccine, but its mechanism on inducing protective immunity is not clear. Previous studies found that the EIAV vaccine strain EIAV_FDDV12 transmembrane protein (gp45) had a high-frequent translational terminating mutation at the site of 261W, resulting in a truncation of 154 amino acid residues at the C-terminus. To explore the biological meaning of the gp45 truncation, a gp45-truncated molecular clone was constructed by using an infectious clone of EIAV_FDDV12 as the backbone. Replications features of the gp45-truncated EIAV and its prototype virus were analyzed and compared in cultivated monocyte-derived macrophages (MDM) of equine and donkey and fetal donkey dermal cells (FDD). Results showed that the replication capacity of the gp45-truncated EIAV in equine and donkey MDMs was significantly decreased compared to the untruncated gp45 EIAV(P < 0.01), especially in the horse macrophages. In contrast, the truncated EIAV replicated significantly faster than the untruncated EIAV in FDD cells (P < 0.01). In addition, the reduced replication of the gp45-truncated EIAV in equine MDM led to a significant decline of cytotoxicity of the host cells when compared with the gp45 untruncated EIAV (P < 0.05). These results suggest that the truncation of the 154-residue C-terminus of the EIAV gp45 glycoprotein was an adaptation to the attenuation of the EIAV vaccine strain in FDD cells. This truncated mutation reduces the replication of the vaccine strain in macrophages, the primary in vivo target cell of EIAV, which leads to a further attenuation of the vaccine.