[Objective] Infectious bovine rhinotracheitis (IBR) is caused by BoHV-1 and there is no commercialized marker vaccine against IBR in China. Based on the previously constructed BoHV-1 gG?/tk? gene-deleted vaccine in this lab, the glycoprotein gD extracellular region sequence which served as the best immunogenicity in BoHV-1 and BoHV-5 was further inserted into the site where tk is located and recombinant virus strains BoHV-1 gG?/tk?/gD+ and BoHV-1 gG?/tk?/gD5+ were successfully constrcuted. This paper aimed to evaluate the immunogenicity and safety of recombinant virus strains in rabbits and protection against both BoHV-1 and BoHV-5 for development of more effective vaccines against BoHV-1 and BoHV-5 infection. [Methods] The 30 rabbits were divided into 6 groups and vaccinated and challenged by nasal inoculation. After strains were vaccinated, their safety was evaluated by clinical symptoms, temperature, virus shedding. Then the rabbits were challenged with either wt BoHV-1 or wt BoHV-5 at the 28th day after vaccination. The protection was evaluated by clinical signs, temperature, virus shedding, histopathology, isolation and identification of virus. Besides, the immunogenicity was studied by neutralization test, indirect-ELISA and specific PBMC proliferative response. [Results] The rabbits vaccinated with BoHV-1 gG?/tk?/gD+ and BoHV-1 gG?/tk?/gD5+ didn’t show clinical signs, nasal virus shedding and viral survival in lung tissues. One rabbit vaccinated with BoHV-1 gG?/tk? had viral survival in lung tissues. After challenge, both strains BoHV-1 gG?/tk?/gD+ and BoHV-1 gG?/tk?/gD5+ could diminish the clinical signs and nasal virus shedding and maintain the good structure of lung tissues indicating the protection against BoHV-1 and cross-protection against BoHV-5 were improved. Besides, BoHV-1 gG?/tk?/gD+ and BoHV-1 gG?/tk?/gD5+ induced stronger neutralization and ELISA antibodies and PBMC proliferation compared with BoHV-1 gG?/tk?. [Conclusion] BoHV-1 gG?/tk?/gD+ and BoHV-1 gG?/tk?/gD5+ are safe and could induce higher levels of immune response than BoHV-1 gG?/tk? strain.