Background Tuberculous spondylitis is a disease that occurs throughout the world. The diagnosis can usually only be made at an advanced stage with severe spinal deformity and significant neurological deficits such as paraplegia. Due to chronic inflammation in bone, there will be an increase in cellular immune response and the release of inflammatory cytokines, which will eventually trigger an increase in the expression of matrix metalloproteinases (MMP). In vitro studies, doxycyline inhibited MMP secretion induced by TB infection. This study assessed the effect of doxycycline on MMP-9 expression in rabbits exposed to tuberculous spondylitis by immunohistochemical examination. Methods This randomized controlled trial study was carried out from January to June 2020. Adult New Zealand rabbits (Oryctolagus cuniculus) were used in this study. A total of 40 rabbits were included in this study. The inoculation of Mycobacterium tuberculosis procedure was carried out in groups. Doxycycline was administered to the control group, the group with a dose of 1 mg/kgBW/day, and the group with a dose of 5 mg/kgBW/day for each group based on time. . The outcome of this study was the examination of the IHC expression of MMP-9 from blood samples after doxycycline administration. Result Forty rabbits inoculated with Mycobacterium tuberculosis and treated with doxycycline for 4 weeks were incubated in individual cages in one large room. The rabbit's appetite does not decrease; the rabbits can defecate and urinate normally, remain active, and respond to the environment well. Four rabbits died during the experiment. There was no significant differences in MMP-9 expression between the control group and intervention group at 2 and 4 weeks. Meanwhile, MMP-9 expression was found greater at 6 and 8 weeks during doxycycline administration between the control and intervention groups. Conclusion Based on the results of this study, there is a positive relationship and effect between doxycycline administration and MMP-9 expression based on immunohistochemical examination of rabbits inoculated for 6 and 8 weeks.