Introduction: Clostridium difficile is an anaerobic spore-forming bacterium recognised as a major cause of nosocomial colitis and antibiotic associated diarrhea. Over the past few years, several studies have focused on the possible role of animals and food as contamination routes for human C. difficile infections. Purpose: The aim of this study was to isolate and compare the C. difficile strains circulating in animals, food and humans in Belgium. Methods: Faecal samples of newborn pigs and calves were collected from breeding farms. Intestinal contents and carcasses samples were collected from cattle and pigs at slaughterhouse. Raw meat was obtained from the retail trade. Horse faecal samples were collected from hospitalized animals. Human C. difficile isolates were obtained from care home residents and hospitalized patients. C. difficile strains were compared with respect to the toxin gene profile, PCR-ribotyping, antimicrobial activity, multilocus sequence typing (MLST) and multiple-locus variable number tandem repeat analysis (MLVA). A neighbourd-joining phylogenetic three was constructed in order to determine the correlation between human, animal and food isolates. Results: A total of 127 isolates belonging to 32 different PCR-ribotypes were collected. The PCR-ribotypes most prevalent in terms of number of isolates were 078, 014 and 027. For a given PCR-ribotype, strains presented a similar susceptibility to the antimicrobials tested, irrespective of the isolation source. Phylogenetic analysis showed that human, meat and animal isolates with the same PCR-ribotype cluster in the same lineage. Significance: The overlap between strains from animal, food and human origins suggest a potential risk of interspecies and foodborne transmission.