AbstractThanks to its ability to isolate previously uncultured bacterial species, culturomics has dynamized the study of the human microbiota. A new bacterial species, Gemella massiliensis Marseille-P3249^T, was isolated from a sputum sample of a healthy French man. Strain Marseille-P3249^T is a facultative anaerobe, catalase-negative, Gram positive, coccus, and unable to sporulate. The major fatty acids were C_16:0 (34%), C_18:1n9 (28%), C_18:0 (15%) and C_18:2n6 (13%). Its 16S rRNA sequence exhibits a 98.3% sequence similarity with Gemella bergeri strain 617-93^T, its phylogenetically closest species with standing in nomenclature. Its digital DNA–DNA hybridization (dDDH) and OrthoANI values with G. bergeri of only 59.7 ± 5.6% and 94.8%, respectively. These values are lower than the thresholds for species delineation (> 70% and > 95%, respectively). This strain grows optimally at 37 °C and its genome is 1.80 Mbp long with a 30.5 mol% G + C content. Based on these results, we propose the creation of the new species Gemella massilienis sp. nov., strain Marseille-P3249^T (= CSUR P3249 = DSMZ 103940).