Despite empirical clinical association of infection with Clostridium difficile with colitis in horses, a causal link has not been confirmed. The objective of this study was to develop a model of C difficile–associated diarrhea in foals with normal transfer of passive immunity. Nine 1-day-old pony foals were inoculated intragastrically with spores or vegetative cells of C difficile. Five foals were challenged with spores, with 2 receiving 105 colony-forming units (CFUs) and concurrently 3 receiving 107 CFUs once daily for 3 days. Clindamycin was administered orally to disrupt gastrointestinal flora. A further 4 foals were challenged by orogastric administration of 1010 CFUs of vegetative cells once daily for 3 days or until diarrhea developed. This group did not receive clindamycin. Spore and vegetative cell preparations were negative for toxins of C difficile and common enteropathogens. Clinical signs varied from mild abdominal discomfort and pasty feces to colic and watery diarrhea in 8 of 9 foals. Four of 5 foals challenged with spores developed mild diarrhea, whereas all foals challenged with vegetative cells developed moderate to severe diarrhea. C difficile was isolated from feces of all foals between 24 and 72 hours after inoculation and toxins A or B or both were detected in the feces of all foals by an enzyme-linked immunosorbent assay. We concluded that spores and vegetative cells of C difficile are capable of colonizing the gastrointestinal tract, producing toxins, and inducing clinical signs similar to those encountered in naturally occurring cases. This study fulfilled Koch's postulates for C difficile–associated diarrhea in foals and provides a model for consistent reproduction of the disease for future studies.