In addition to their use in cheese technology, dairy propionibacteria have been identified as potential probiotics. However, to have a probiotic effect, propionibacteria have to survive and to remain metabolically active in the digestive tract. The aim of the present study was to investigate the survival and metabolic activity of Propionibacterium freudenreichii within the gastrointestinal tract of human microbiota-associated rats, and its influence on intestinal microbiota composition and metabolism. Twenty-five dairy Propionibacterium strains were screened for their tolerance towards digestive stresses and their ability to produce propionate in a medium mimicking the content of the human colon. Three strains were selected and a daily dose of 2 × 1010 colony-forming units was fed to groups of human microbiota-associated rats for 20 d before microbiological, biochemical and molecular investigations being carried out. These strains all reached 8-log values per g faeces, showing their ability to survive in the gastrointestinal tract. Transcriptional activity within the intestine was demonstrated by the presence of P. freudenreichii-specific transcarboxylase mRNA. The probiotic efficacy of propionibacteria was yet species- and strain-dependent. Indeed, two of the strains, namely TL133 and TL1348, altered the faecal microbiota composition, TL133 also increasing the caecal concentration of acetate, propionate and butyrate, while the third strain, TL3, did not have similar effects. Such alterations may have an impact on gut health and will thus be taken into consideration for further in vivo investigations on probiotic potentialities of P. freudenreichii.