The RD1 genomic region is present in virulent strains of Mycobacterium tuberculosis (MTB), missing from the vaccine strain M. bovis BCG, and its importance to virulence has been established experimentally. Based on in silico analysis, it has been suggested that RD1 may encode a novel secretion system, but the mechanism by which this region affects virulence is unknown. Here we examined mutants disrupted in five individual RD1 genes. Both in vitro and in vivo, each mutant displayed an attenuated phenotype very similar to a mutant missing the entire RD1 region. Genetic complementation of individual genes restored virulence. Attenuated mutants could multiply within THP-1 cells, but they were unable to spread to uninfected macrophages. We also examined export of two immunodominant RD1 proteins, CFP-10 and ESAT-6. Export of these proteins was greatly reduced or abolished in each attenuated mutant. Again, genetic complementation restored a wild-type phenotype. Our results indicate that RD1 genes work together to form a single virulence determinant, and argue that RD1 encodes a novel specialized secretion system that is required for pathogenesis of MTB.