Tuberculosis (TB) is a major cause of morbidity and mortality worldwide. This study evaluated the immune response in mice to members of the Mtb9.9 protein family. C57BL/6 mice were immunized with four different Mtb9.9 recombinant proteins to evaluate the humoral and cellular immune responses to each protein. Mouse splenocytes from these mice were stimulated in vitro for 72 h with the Mtb9.9 proteins. Concentrations of the cytokines interferon (IFN)-γ and tumor necrosis α (TNF-α) in the culture supernatants were increased compared with controls. Sera were taken from the same mice at 3 weeks after immunization and contained significantly higher levels of immunoglobulin (Ig)G2c antibody when compared with sera from control mice. Our findings indicate a bias toward T helper (Th)1-type cellular and humoral memory responses against Mtb9.9 recombinant proteins in mice. This study demonstrates that these proteins might be suitable candidates for use as subunit vaccines.