MicroRNAs (miRNAs) play an important role in the regulation of the immune system. To demonstrate the role of miR-125b-5p in anti-TB immunity, we have infected host target lung alveolar A549, human macrophage THP-1, mice macrophage RAW264.7 and C57BL/6 mice, respectively. The q-PCR results demonstrated that miR-125b-5p is highly expressed in the tested conditions. The expression of miR-125b-5p in BCG infected mice lung was about 15 times of that in the control mice. The results verified that miR-125b-5p regulated mycobacteria growth in A549, THP-1 and RAW264.7 cells, respectively. Moreover, administration of miR-125b-5p inhibitors in BCG infected mice significantly reduced bacteria burden in lung. Thus, we concluded that miR-125b-5p may play a critical role on mycobacteria proliferation in vivo. The results provide useful information for our understanding of host immune defense and potential therapeutic interventions against this disease.