The only licensed vaccine Mycobacterium bovis Bacillus Calmette-Guérin (BCG) cannot prevent the prevalence of tuberculosis (TB), which remains a major public health problem worldwide. A more effective TB vaccine than BCG is urgently needed. Subunit vaccine is a promising strategy, and suitable adjuvants will benefit the development of effective TB subunit vaccines. MTO, consisting of monophosphoryl lipid A (MPLA), trehalose-6,6'-dibehenate (TDB), and MF59, was developed as an adjuvant of TB vaccine because of its ability to evoke the Th1-type T cell responses, while it is insufficient to induce single and multifunctional IL-2(+) T cells and has a limited ability to confer protection against Mycobacterium tuberculosis infection. Heat-killed Mycobacterium vaccae (Mv), which can evoke cytotoxic CD8(+) and CD4(+) T cell responses and has adjuvanticity, was, in this study, combined with MTO to produce a new adjuvant, called MTOM. The TB fusion protein Rv3407-PhoY2-Ag85A-Rv2626c-RpfB (WH121) was mixed with MTO, Mv, and MTOM to produce three subunit vaccines, and the protective efficacy and immune responses were compared in C57BL/6 mice. WH121/MTOM provided better protection against TB than the other two vaccines, matching the performance of BCG vaccine. MTOM showed stronger ability to increase single and multifunctional IL-2(+) T cells and induce Th1-type responses than MTO or Mv. Therefore, MTOM might be a promising adjuvant that could contribute to the development of TB subunit vaccines.