The koala retrovirus (KoRV) is spreading in the koala population from the north to the south of Australia and is also in the process of endogenization into the koala genome. Virus infection is associated with tumorigenesis and immunodeficiency and is contributing to the decline of the animal population. Antibody production is an excellent marker of retrovirus infection; however, animals carrying endogenous KoRV are tolerant. Therefore, the therapeutic immunization of animals carrying endogenous KoRV seems to be ineffective. Using the recombinant transmembrane (TM) envelope protein of the KoRV, we immunized goats, rats and mice, obtaining in all cases neutralizing antibodies which recognize epitopes in the fusion peptide proximal region (FPPR), and in the membrane-proximal external region (MPER). Immunizing several animal species with the corresponding TM envelope protein of the closely related porcine endogenous retrovirus (PERV), as well as the feline leukemia virus (FeLV), we also induced neutralizing antibodies with similar epitopes. Immunizing with the TM envelope protein in addition to the surface envelope proteins of all three viruses resulted in higher titers of neutralizing antibodies. Immunizing KoRV-negative koalas with our vaccine (which is composed of both envelope proteins) may protect these animals from infection, and these may be the starting points of a virus-free population.