Clostridium difficile is the leading cause of infectious nosocomial diarrhea. The pathogenesis of C. difficile infection (CDI) results from the interactions between the pathogen, intestinal epithelium, host immune system, and gastrointestinal microbiota. Previous studies of the host-pathogen interaction in CDI have utilized either simple cell monolayers or in vivo models. While much has been learned by utilizing these approaches, little is known about the direct interaction of the bacterium with a complex host epithelium. Here, we asked if human intestinal organoids (HIOs), which are derived from pluripotent stem cells and demonstrate small intestinal morphology and physiology, could be used to study the pathogenesis of the obligate anaerobe C. difficile. Vegetative C. difficile, microinjected into the lumen of HIOs, persisted in a viable state for up to 12 h. Upon colonization with C. difficile VPI 10463, the HIO epithelium is markedly disrupted, resulting in the loss of paracellular barrier function. Since similar effects were not observed when HIOs were colonized with the nontoxigenic C. difficile strain F200, we directly tested the role of toxin using TcdA and TcdB purified from VPI 10463. We show that the injection of TcdA replicates the disruption of the epithelial barrier function and structure observed in HIOs colonized with viable C. difficile.