Background: Tuberculosis is a leading cause of death worldwide. BCG is an effective vaccine, but not widely used in many parts of the world due to a variety of issues. Mycobacterium vaccae (M. vaccae) is another vaccine used in human subjects to prevent tuberculosis. In the current study, we investigated the potential mechanisms of M. vaccae vaccination by determining differentially expressed genes in mice infected with M. tuberculosis before and after M. vaccae vaccination.Methods: Three days after exposure to M. tuberculosis H37Rv strain (5×105 CFU), adult BALB/c mice randomly received either M. vaccae vaccine (22.5 μg) or vehicle via intramuscular injection (n=8). Booster immunization was conducted 14 and 28 days after the primary immunization. Differentially expressed genes were identified by microarray followed by standard bioinformatics analysis. Results: M. vaccae vaccination provided protection against M. tuberculosis infection (most prominent in the lungs). We identified 2,326 upregulated and 2,221 downregulated genes in vaccinated mice. These changes could be mapped to a total of 123 signaling pathways (68 upregulated and 55 downregulated). Further analysis pinpointed to the MyD88-dependent TLR signaling pathway and PI3K-Akt signaling pathway as most likely to be functional. Conclusions: M. vaccae vaccine provided good protection in mice against M. tuberculosis infection, via a highly complex set of molecular changes. Our findings may provide clue to guide development of more effective vaccine against tuberculosis.