To investigate essential factors that determine the efficacy of vaccines against lentiviruses, an effective attenuated equine infectious anemia virus (EIAV) vaccine strain and a proviral derivative of the vaccine were compared with respect to differences in inducing protective immunity. Although these two strains replicated equally well in vitro and in vivo, the proviral strain induced significantly less protection from disease and infection caused by viral challenge and significantly lower specific neutralizing capability. These findings indicated that the proviral strain had lost the ability to stimulate immune protection compared to the parental vaccine strain. A further analysis of the envelope gp90 gene variation revealed that compared to the proviral strain, the vaccine strain displayed a wide sequence diversity in immunogen composition. Thus, we inferred that the differences in immunogen composition might be the major cause for the failure of the proviral derivative to elicit the immune protection induced by the parental strain.