<p><i>Mycobacterium tuberculosis</i> (Mtb), a highly infectious intracellular pathogen, is the primary causative agent of tuberculosis (TB), which ranks as the leading cause of death from individual infectious agents. In this context, we developed a model using macrophages infected with <i>M. smegmatis</i> mc2155 and Mtb H37Ra to elucidate the role of Mn²⁺ in host defense against tuberculosis infection. Treatment with a combination of anti-tuberculosis drugs and Mn²⁺ for 36&#xa0;h markedly reduced the survival of intracellular bacteria, and the addition of Mn²⁺ significantly enhanced the secretion of TNF-α and IL-1β. In mice infected with Mtb H37Ra, treatment with Mn²⁺ (2&#xa0;mg/kg) and the combination of Isoniazid and Mn²⁺ significantly increased serum TNF-α levels, with the combination treatment showing a more pronounced effect. Histopathological analysis indicated a relatively diminished immune response in the spleen in the drug and Mn²⁺treatment groups, while the immune response in the lungs was comparable across all groups. Mn²⁺ can synergistically enhance the efficacy of anti-tuberculosis drugs by activating cellular secretion of immune factors and inhibiting the growth of Mtb. The findings of this study provide a rationale for the potential use of Mn²⁺ as an activator of cellular immunity against Mtb infection.</p>