Tuberculosis (TB) is currently the leading cause of death in humans by a single infectious agent, Mycobacterium tuberculosis. The Bacillus Calmette-Guérin (BCG) vaccine prevents pulmonary TB with variable efficacy, but can cause life-threatening systemic infection in HIV-infected infants. In this study, TBvac85, a derivative of Mycobacterium shottsii expressing M. tuberculosis Antigen 85B, was examined as a safer alternative to BCG. Intranasal vaccination of guinea pigs with TBvac85, a naturally temperature-restricted species, resulted in serum Ag85B-specific IgG antibodies. Delivery of the vaccine by this route also induced protection equivalent to intradermal BCG based on organ bacterial burdens and lung pathology six weeks after aerosol challenge with M. tuberculosis strain Erdman. These results support the potential of TBvac85 as the basis of an effective TB vaccine. Next-generation derivatives expressing multiple M. tuberculosis immunogens are in development.