Direct Mycobacterium tuberculosis inoculation on rabbit vertebral body was used in rabbit spinal infection study. The potential spread of Mycobacterium tuberculosis into the environment will be observed in order to create the conditions fulfilling biosafety aspects. Two groups of six New Zealand rabbits were treatment group (n=4) and control group (n=2). The treatment group had injection of 0.1?mL (107?cfu/mL) suspension of Mycobacterium tuberculosis into the vertebral body T12. They were incubated for 2 to 14 weeks. One rabbit per period of 2, 4, 6, and 14 weeks was euthanized to collect feces, urine, saliva, and tissue lesions. The control group had only feces, urine, and saliva to detect bacteria using AFB staining, culture, and PCR. Both two groups were kept in individual cages. They were put together in a large cage for 3 hours every day to interact with each other. AFB staining, culture, and radiological examination showed negative result, but in one rabbit, histopathological examination showed positive result and PCR examination in another rabbit of the treatment group. Spreading score was 1.05% and infected score was 0 (null). The procedure did not reveal the potential spread of Mycobacterium tuberculosis into the environment.