Objective To study the regulatory effect and mechanism of silencing microRNA-16 on immune function in mice with pulmonary tuberculosis. Methods Forty CD2F1 female mice were divided into the control group, model group, overexpression group, and silent group (n = 10 per group). In the model group, overexpression group, and silent group, the pulmonary tuberculosis model was established. After successful modeling, the overexpression group and silent group were aseptically injected with 10 μL microRNA-16 lentivirus suspension. T lymphocyte subsets, thymus index, lung inflammatory factors and toll like receptor (TLRs) signaling pathway factors were detected in lung tissues. Results The levels of CD3+, CD4+, CD4+ / CD8+, thymus index and IL-10 in the overexpression group were lower than those in the model group, while the levels of CD8+, IL-6, TNF-α, TLR2, TLR4 and NF-κB in the overexpression group were higher than those in the model group (P < 0. 05).The levels of CD3+, CD4+, CD4+ / CD8+, thymus index and IL-10 in the silence group were higher than those in the model group, while the levels of CD8+, IL-6, TNF-α, TLR2, TLR4 and NF-κB in the silence group were lower than those in the model group (P < 0. 05).The levels of CD3+, CD4+, CD4+/ CD8+, thymus index and IL-10 in the silence group were higher than those in the over expression group, and the levels of CD8+, IL-6, TNF-α, TLR2, TLR4 and NF-κB were lower than those in the over expression group (P < 0. 05). Conclusions Silencing microRNA-16 may play a role in regulating the immune function in mice with tuberculosis, by acting on TLR signaling pathways and inhibiting abnormal expression of pathway factors, thus regulating the immune response and improving immune disorders.