Tuberculosis (TB) is currently the leading cause of death among bacterial infectious diseases. The spectrum of disease manifestations depends on both host immune responses and the ability of Mycobacterium tuberculosis to resist it. Small non-coding RNAs are known to regulate gene expression; however, their functional role in the relationship of M. tuberculosis with the host is poorly understood. Here, we investigated the effect of small non-coding sRNAs MTS1338 and MTS0997 on M. tuberculosis properties by creating knockout strains. We also assessed the effect of small non-coding RNAs on the survival of wild type and mutant mycobacteria in primary cultures of human alveolar macrophages and the virulence of these strains in a mouse infection model. Wild-type and mutants survived differentially in human alveolar macrophages. Infection of I/St mice with KO M. tuberculosis H37RV strains provided beneficial effects onto major TB phenotypes. We observed attenuated tuberculosis-specific inflammatory responses, including reduced cellular infiltration and decreased granuloma formation in the lungs. Infections caused by KO strains were characterized by significantly lower inflammation of mouse lung tissue and increased survival time of infected animals. Thus, the deletion of MTS0997 and MTS1338 lead to a significant decrease in the virulence of M. tuberculosis.