In Europe, the Gram-positive spore-forming anaerobic bacterium Clostridium difficile (CD) is the leading cause of intestinal nosocomial post-antibiotic infections in adults. The CD infections (CDI) generally occur in patients with compromised gut microbiota. CD is responsible for 15-25% of cases of antibiotic-associated-diarrhoea and for almost all pseudomembranous colitis cases. CDI is a multi-step process that includes i) oral infection by environmental spores of toxigenic CD strains or caught off hospital staff, ii) spore germination in dysbiotic intestinal lumen, iii) gut colonization by CD vegetative cells (VC) and iv) production of toxins responsible for the symptoms followed by the dissemination of CD spores. The virulence of CD is primarily due to the production of two cytolytic toxins: TcdA and TcdB, which exert deleterious effects on epithelial cells. However many virulence factors seem to be involved during the gut colonization and the process of CDI. Therefore, the aims of our research were to decipher the key steps of the CD pathogenesis and particularly the intestinal colonization, which is an essential prerequisite before toxin production. This include i) the adaptive strategies used by CD in response to stresses encountered during infection including mechanisms of resistance to oxidative/nitrosative stress and biofilm formation; ii) the complex developmental sporulation process and the function of proteins associated to the spore surface and their involvement in the morphology and the protection/signaling/ adhesion of the spores; iii) the RNA-based mechanisms controlling CDI steps involving the c-di-GMP/AMP signaling pathway; iv) the mechanisms of the regulation of toxin production in relation to cellular metabolic pathways and v) the impact of prophages in the genetic variability of CD strains.