In this study, we investigated the residual virulence of mutants of Mycobacterium tuberculosis that are defective in 4 of the 5 rpf-like genes, their capacity to persist in the murine host and the utility present in these mutants to serve as novel vaccine candidates. Our data indicate that the two quadruple rpf deletion mutants, ΔACBD and ΔACDE, both display significant attenuation in the mouse lungs after aerosol infection, with no observable increase in bacillary loads upon aminoguanidine-induced immune suppression. However, after subcutaneous injection these strains were able to persist at the low level, similar to that of BCG, in the mouse lungs and lymphoid organs. Furthermore, both rpf quadruple mutants were able to enhance the numbers of IFN-γ-producing T-cells in spleens to a level comparable to that of BCG, and conferred protection upon subsequent challenge with virulent M. tuberculosis in terms of mycobacterial multiplication in organs and survival time. The reduction in organ bacillary loads after vaccination with ΔACDE was comparable to that of BCG, while ΔACBD displayed a small but statistically significant enhancement in protection compared to BCG. Collectively, these data suggest that rpf deletion mutants show potential for further development as novel vaccine candidates for tuberculosis.