Clostridium difficile is responsible for a large spectrum of intestinal diseases ranging from mild diarrhea to fatal colitis depending on the one hand on the strain virulence and on the other on the host. The pathogenesis of C. difficile infection could be seen as a three-step process that takes place after disruption of the digestive microbiota by antibiotics: (1) contamination by and germination of spores; (2) multiplication of vegetative cells in the colonic niche using colonization factors; (3) production of the two toxins TcdA and TcdB and for some strains an additional toxin, the binary toxin CDT. Several studies have been performed to characterize the bacterial factors involved in the colonization step and particularly adhesins.Here, we describe first the methods used to study C. difficile adherence in vitro to epithelial cells and in vivo in animal model intestinal tract, and second the methods used to demonstrate the adhesive properties of surface proteins using Cwp66, GroEL, and FbpA as examples.