“Clostridium neonatale”was recently described as a new species within the Cluster I of theClostridiumgenussensu stricto. In this study, we characterized“C. neonatale”isolates (n¼42) and compared theirphenotypic properties with those ofClostridium butyricum(n¼26), a close related species. Strainsisolated from fecal samples of healthy neonates were tested for different phenotypic characteristics.Compared toC. butyricum,“C. neonatale”showed a significant higher surface hydrophobicity(p¼0.0047), exopolysaccharide production (p¼0.0069), aero-tolerance (p¼0.0222) and viability at30C(p¼0.0006). A lower swimming ability (p¼0.0146) and tolerance against bile (0.3%) (p¼0.0494),acid (pH 4.5) (p<0.0001), osmolarity (NaCl 5%,p¼0.0188) and temperature at 50C(p¼0.0013)characterized“C. neonatale”strains. Our results showed that“C. neonatale”behaves very differently fromC. butyricumand suggests specific responses to environmental changes. Besides it is thefirst study onclinical isolates for these two anaerobic members of the newborns' gut microbiota and broadens ourknowledge about their phenotypic traits“Clostridium neonatale”was recently described as a new species within the Cluster I of theClostridiumgenussensu stricto. In this study, we characterized“C. neonatale”isolates (n¼42) and compared theirphenotypic properties with those ofClostridium butyricum(n¼26), a close related species. Strainsisolated from fecal samples of healthy neonates were tested for different phenotypic characteristics.Compared toC. butyricum,“C. neonatale”showed a significant higher surface hydrophobicity(p¼0.0047), exopolysaccharide production (p¼0.0069), aero-tolerance (p¼0.0222) and viability at30C(p¼0.0006). A lower swimming ability (p¼0.0146) and tolerance against bile (0.3%) (p¼0.0494),acid (pH 4.5) (p<0.0001), osmolarity (NaCl 5%,p¼0.0188) and temperature at 50C(p¼0.0013)characterized“C. neonatale”strains. Our results showed that“C. neonatale”behaves very differently fromC. butyricumand suggests specific responses to environmental changes. Besides it is thefirst study onclinical isolates for these two anaerobic members of the newborns' gut microbiota and broadens ourknowledge about their phenotypic traits