Seven bifidobacterial strains were isolated from the faeces of two adult males of the two-toed sloth (Choloepus didactylus) housed in Parco Natura Viva, in Italy. Comparative sequence analysis of 16S rRNA and of five housekeeping (hsp60, rpoB, clpC, dnaJ, dnaG) genes revealed that these strains were classified into two clusters. On the basis of 16S rRNA gene sequence similarity, the type strain of Bifidobacterium catenulatum subsp. kashiwanohense DSM 21854^T (95.4 %) was the closest neighbour to strain in Cluster I (BRDM 6^T), whereas the type strain of Bifidobacterium dentium DSM 20436^T (values were in the range of 98‒99.8 %) was the closest neighbour to the other six strains in Cluster II. The average nucleotide identity (ANI) values of BRDM 6^T and of strains in Cluster II with the closely related type strains were 76.0 and 98.9 % (mean value) respectively. Therefore, genotyping based on the genome sequence of the strain BRDM 6^T combined with phenotypic analyses clearly revealed that the strain BRDM 6^T represents a novel species for which the names Bifidobacterium choloepi sp. nov. (BRDM 6^T=NBRC 114053^T=BCRC 81222^T) is proposed.