Abstract Background H56:IC31 is a candidate tuberculosis vaccine comprising a fusion protein of Ag85B, ESAT-6 and Rv2660c, formulated in IC31 adjuvant. This first-in-human, open label phase I trial assessed the safety and immunogenicity of H56:IC31 in healthy adults without or with Mycobacterium tuberculosis ( M.tb ) infection. Methods Low dose (15 μg H56 protein in 500 nmol IC31) or high dose (50 μg H56, 500 nmol IC31) vaccine was administered intramuscularly thrice, at 56-day intervals. Antigen-specific T cell responses were measured by intracellular cytokine staining and antibody responses by ELISA. Results One hundred and twenty-six subjects were screened and 25 enrolled and vaccinated. No serious adverse events were reported. Nine subjects (36%) presented with transient cardiovascular adverse events. The H56:IC31 vaccine induced antigen-specific IgG responses and Th1 cytokine-expressing CD4+ T cells. M.tb -infected vaccinees had higher frequencies of H56-induced CD4+ T cells than uninfected vaccinees. Low dose vaccination induced more polyfunctional (IFN-γ+ TNF-α+ IL-2+ ) and higher frequencies of H56-specific CD4+ T cells compared with high dose vaccination. A striking increase in IFN-γ-only-expressing CD4+ T cells, displaying a CD45RA− CCR7− effector memory phenotype, emerged after the second high-dose vaccination in M.tb -infected vaccinees. TNF-α+ IL-2+ H56-specific memory CD4+ T cells were detected mostly after low-dose H56 vaccination in M.tb -infected vaccinees, and predominantly expressed a CD45RA− CCR7+ central memory phenotype. Our results support further clinical testing of H56:IC31.