Various new tuberculosis (TB) vaccine candidates in combination with new delivery systems, including subunit vaccines, are currently being evaluated by a number of laboratories. One vaccine candidate that has shown promising protective capacity in mice and guinea pigs is a fusion of Ag85B and ESAT-6. In this study, we have investigated the efficacy of this Ag85B–ESAT-6 fusion protein vaccine in a non-human primate model for TB. Vaccination of cynomolgus monkeys with the Ag85B–ESAT-6 fusion protein in two different adjuvant (DDA/MPL, AS02A) resulted in a reduction in bacterial number and/or lung pathology in animals challenged with Mycobacterium tuberculosis. Vaccination prevented an increase in C-reactive protein serum levels, general activation of CD4 and CD8 subsets and boosted development of humoral and cellular immune responses to a spectrum of mycobacterial antigens on exposure to M. tuberculosis infection. We show, in two independent experiments, that vaccination of primates with Ag85B–ESAT-6 induces protective immune responses, suggesting that Ag85B–ESAT-6 is a strong candidate for further clinical evaluation. As far as we are aware this is the first report of protection in primates with a subunit vaccine.