Tuberculosis (TB) remains a staggering burden on global public health. Novel preventative tools are desperately needed to reach the targets of the WHO post-2015 End-TB Strategy. Peptide or protein-based subunit vaccines offer potential as safe and effective generators of protection， and enhancement of local pulmonary immunity may be achieved by mucosal delivery. We describe the synthesis of a novel subunit vaccine via native chemical ligation. Two immunogenic epitopes， ESAT6 and TB10.4 from (Mtb)， were covalently conjugated to the TLR2-ligand PamCys to generate a self-adjuvanting lipopeptide vaccine. When administered mucosally to mice， the vaccine enhanced pulmonary immunogenicity， inducing strong Th17 responses in the lungs and multifunctional peripheral T-lymphocytes. Mucosal， but not peripheral vaccination， provided substantial protection against Mtb infection， emphasizing the importance of delivery route for optimal efficacy.