Nontuberculous Mycobacteria (NTM) respiratory infections have been gradually increasing. Here, THP-1 cells were used as a model to evaluate intracellular persistence of three NTM species (reference and clinical strains) in human alveolar macrophages. The contribution of phagosome acidification, nitric oxide (NO) production and cell dead on NTM intracellular fate was assessed. In addition, strains were characterized regarding their repertoire of virulence factors by whole-genome sequencing. NTM experienced different intracellular fates: M. smegmatis and M. fortuitum ATCC 6841 were cleared within 24h. In contrast, M. avium strains (reference/clinical) and M. fortuitum clinical strain were able to replicate. Despite this fact, unexpectedly high percentages of acidified phagosomes were found harbouring rab7, but not CD63. All NTM were able to survive in vitro at acidic pHs, with the exception of M. smegmatis. Our data further suggested a minor role for NO in intracellular persistence and that apoptosis mediated by caspase 8 and 3/7, but not necrosis, is triggered during NTM infection. Insights regarding the bacteria genomic backbone corroborated the virulence potential of M. avium and M. fortuitum. In conclusion, the phenotypic traits detected contrast with those described for M. tuberculosis, pointing out that NTM adopt distinct strategies to manipulate the host immune defense and persist intracellularly.Keywords: Nontuberculous Mycobacteria (NTM); host-pathogen interaction; macrophages; pH; virulence.