The aim of the study was to evaluate in vitro the potential probiotic functionality in the gut of animal-originated bifidobacteria strains. Eleven B. animalis and five B. pseudolongum strains isolated from Wistar rats were characterized for their tolerance to low pH and bile, and seven B. animalis and four B. pseudolongum strains for their ability to adhere to HT-29 cells and mucus. The strains showed high resistance to three-hour exposure to pH 3 (34-100% of the population survived) and six-hour exposure to the presence of 3% bile (28-93%), as well as differentiated adhesive abilities. The number of bacterial cells attached to 100 HT-29 cells ranged from 9 to 33 and from 17 to 645 for B. pseudolongum and B. animalis strains, respectively, whereas corresponding levels of adherence per square centimetre of polystyrene-immobilized mucin ranged from 3 × 10⁵ to 4 × 10⁵ and from 5 × 10⁵ to 889 × 10⁵ bacterial cells. Three B. animalis strains (KSP4, PS46 and PS11) resistant to low pH and bile, well-adhering to epithelial cells and mucus were selected as potential probiotics for animals and will be evaluated in future in vivo studies.