The continuing emergence of new strains of antibiotic-resistant bacteria has renewed interest in phage therapy; however, there has been limited progress in applying phage therapy to multi-drug resistant Mycobacterium tuberculosis (Mtb) infections. In this study, we tested three bacteriophage strains for their Mtb-killing activities and found that two of them efficiently lysed Mtb H37Rv in 7H10 agar plates. However, only phage DS6A efficiently killed H37Rv in liquid culture and in Mtb-infected human primary macrophages. In subsequent experiments, we infected humanized mice with aerosolized H37Rv, then treated these mice with DS6A intravenously to test its in vivo efficacy. We found that DS6A treated mice showed increased body weight and improved pulmonary function relative to control mice. Furthermore, DS6A reduced Mtb load in mouse organs with greater efficacy in the spleen. These results demonstrated the feasibility of developing phage therapy as an effective therapeutic against Mtb infection.