Efficient protein-based vaccine delivery systems are needed to achieve a persistent memory immune response capable of detecting and eliminating intracellular pathogens such as Mycobacterium tuberculosis (TB). We have developed a novel protein-microsphere formulation using the recently discovered TB antigen Mtb8.4. Immunization of mice with a single dose of this Mtb8.4-microsphere formulation resulted in both humoral and cellular responses against Mtb8.4. The Mtb8.4-specific CD8 T-cell responses following a single administration of Mtb8.4-microspheres exceeded that elicited by protein plus adjuvant following multiple immunizations. These results demonstrate the efficacy of a single dose protein-microsphere vaccine for the induction of strong cell-mediated and humoral immune responses against M. tuberculosis antigens.