<title>Abstract</title>The global incidence of the human nontuberculous mycobacteria (NTM) disease is rapidly increasing. However, knowledge of gene essentiality under optimal growth conditions and conditions relevant to the natural ecology of NTM, such as hypoxia, is lacking. In this study, we utilized transposon sequencing to comprehensively identify genes essential for growth in <italic>Mycobacterium intracellulare</italic>. Of 5126 genes of <italic>M. intracellulare</italic> ATCC13950, 506 genes were identified as essential genes, of which 280 and 158 genes were shared with essential genes of <italic>M. tuberculosis</italic> and <italic>M. marinum</italic>, respectively. The shared genes included target genes of existing antituberculous drugs including SQ109, which targets the trehalose monomycolate transporter MmpL3. From 175 genes showing decreased fitness as conditionally essential under hypoxia, preferential carbohydrate metabolism including gluconeogenesis, glyoxylate cycle and succinate production was suggested under hypoxia. Virulence-associated genes including proteasome system and mycothiol redox system were also identified as conditionally essential under hypoxia, which was further supported by the higher effective suppression of bacterial growth under hypoxia compared to aerobic conditions in the presence of these inhibitors. This study has comprehensively identified functions essential for growth of <italic>M. intracellulare</italic> under conditions relevant to the host environment. These findings provide critical functional genomic information for drug discovery.