ABSTRACT Rabbit hemorrhagic disease virus 2 and Clostridium perfringens type A cause infections in rabbit. Vaccines are considered an effective strategy for fighting these infections. Nowadays, the demand for using a nanoparticle adjuvant as (Montanide™ IMS) is increased due to its ability for enhancing both humoral and cell mediated immunity and, in addition, it can be administrated through different routes. An inactivated vaccine against rabbit hemorrhagic disease virus 2 and Clostridium perfringens type A which adjuvanted by Montanide™ IMS 1313 N VG PR (IMS 1313) was developed. The prepared vaccine was evaluated in rabbits for sterility, safety and potency via two different routes of vaccination. Oral administration of inactivated vaccine was evaluated as an alternative route to subcutaneous vaccination. The results revealed that rabbits vaccinated by subcutaneous route exhibited satisfactory antibody and antitoxin titer against rabbit hemorrhagic disease virus 2 and Clostridium perfringens type A, respectively, from 2nd week post vaccination and reached the peak at 3th week post vaccination. On the other hand, antibody and antitoxin titer of orally vaccinated rabbits didn't reach the satisfactory level. Rabbits vaccinated orally were not protected against virulent rabbit hemorrhagic disease virus 2, with 30% protection, while rabbits vaccinated subcutaneously showed satisfactory protection (90%). Serum nitric oxide and lysozyme activity had significant differences between vaccinated and control rabbits. The level of nitric oxide and lysozyme in sera of subcutaneously vaccinated rabbits was higher than that of orally vaccinated rabbits. Interleukin-6 and tumor necrosis factor-ɑ were determined in the spleen of vaccinated rabbits, significant differences were obtained between subcutaneously and orally vaccinated rabbits. It was concluded that the combined vaccine is potent when inoculated by subcutaneous route in contrast to the oral route. The Montanide™ IMS 1313 adjuvant is a product that can be used for rabbit vaccine preparation.