The potent IFN-gamma inducing fusion antigen Ag85B-ESAT-6 (85B6) is a lead subunit candidate to improve current vaccination against Mycobacterium tuberculosis (Mtb). The recombinant M. bovis BCG strain Myc3504 was constructed to secrete 85B6. It was based on commercial BCG strain Moreau Rio de Janeiro (BCG(MoWT)) which remains available for human oral administration. Myc 3504 induced higher levels of 85B6-specific IFN-gamma circulating T-cells as compared to BCG(MoWT). A novel needle-free mucosal immunization regimen combining oral prime with Myc3504 or BCG(MoWT) with intranasal boost with LTK-63-adjuvanted 85B6 was compared to subcutaneous prime-boost immunization. Strikingly whereas parenteral immunization induced sustained levels of 85B6-specific IFN-gamma secretion by circulating T-cells, mucosal regimens induced barely detectable IFN-gamma. Despite this, mice and guinea pigs immunized with the mucosal regimens were as efficiently protected against aerosol Mtb challenge as parenterally immunized animals. After Mtb challenge, anti-ESAT-6 IFN-gamma responses sharply increased in non-vaccinated mice as a hallmark of infection. Parenterally immunized mice that controlled Mtb infection, displayed anti-ESAT-6 IFN-gamma responses as high as non-immunized infected mice, compromising the possible use of ESAT-6 as a diagnostic tool. Interestingly, in mucosally immunized mice that were equally protected, post-challenge ESAT-6-specific IFN-gamma T-cell response remained low.