Nonribosomal peptide synthases produce short peptides in a manner that is distinct from classical mRNA-dependent ribosome-mediated translation. The genome harbors a nonribosomal peptide synthase gene, , which is part of a gene cluster proposed to be involved in the biosynthesis of isonitrile lipopeptides. Orthologous clusters are found in other slow-growing pathogenic mycobacteria and actinomycetes. To probe the role of the gene in infection, we generated an deletion mutant in H37Rv and tested its virulence in immunocompetent (C57BL/6) mice. The mutant strain displayed lower initial growth rates in the lungs and a defective dissemination to the spleens of infected mice. Mice infected with the mutant strain also survived for twice as long as those infected with wild-type and, remarkably, showed subdued pathology, despite similar bacterial loads at later stages of infection. The differences in the course of infection between wild-type and mutant strains were accompanied by distinct dynamics of the immune response. Most strikingly, the mutant was highly attenuated in immunodeficient (SCID-, recombination activating 2 [RAG2]-, and gamma interferon [IFN-γ]-deficient) mice, suggesting that macrophages control the mutant more efficiently than they control the wild-type strain. However, in the presence of IFN-γ, both strains were equally controlled. We propose that the gene and its associated cluster are drivers of virulence during the early stages of infection. Over 10 million people developed tuberculosis (TB) in 2016, and over 1.8 million individuals succumbed to the disease. These numbers make TB the ninth leading cause of death worldwide and the leading cause from a single infectious agent. Therefore, finding novel therapeutic targets in , the pathogen that causes most cases of human TB, is critical. In this study, we reveal a novel virulence factor in , the gene. The lack of highly attenuates the course of infection in the mouse model, which is particularly relevant in immune-deficient hosts. This is very relevant as TB is particularly incident in immune-suppressed individuals, such as HIV patients.