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Objective: To identify the risk factors of Clostridium difficile infection (CDI) in diarrheal patients admitted to the intensive care unit (ICU) in Tehran Baharloo Hospital.Methods: A case-control study was conducted on ICU patients with hospital-acquired diarrhea. A total of 101 patients were divided into two groups: the case group (CDI positive, n=47) and the control group (CDI negative, n=54). The baseline information such as use of antibiotics, other drugs administration, treatments before diarrhea, laboratory results, and vital signs of the two groups were compared. Besides, logistic regression model was used to assess the correlation between CDI positivity and mortality. Results: Hospital stay length, ICU stay length, duration from admission to diarrhea onset, and nasogastric feeding duration, mechanical ventilation rate and its duration were significantly different from these of the control group (P<0.05). The frequency of proton pump inhibitor and carbapenem in the case group was significantly higher than that of the control group (P<0.05). In addition, age had a significant effect on the mortality of CDI patients. Conclusions: Patients with older age, longer duration of hospital or ICU stay, longer duration of endotracheal feeding and/or intubation were more susceptible to CDI. In addition, proton pump inhibitor and carbapenem use influenced the gut microbiome diversity and increased the CDI risk in patients.