Clostridium difficile infection (CDI) is an increasing concern in China. However, the risk factors of CDI are rarely reported in the Chinese population. A prospective observational study was therefore conducted among patients with hospital-acquired C. difficile diarrhoea and the risk factors of CDI in a retrospective case-control study. The CDI patients were compared with the non-CDI diarrhoeal patients and those without diarrhoea, respectively. The recurrent CDI patients were compared with the corresponding non-recurrent CDI patients and those without diarrhoea, respectively. Overall, of the 240 patients with hospital-acquired diarrhoea 90 (37.5%) were diagnosed as CDI, and 12 (13.3%) of the 90 CDI patients experienced recurrence. Multivariate analysis indicated that renal disease, malignancy, hypoalbuminemia, prior antibiotic treatment, chemotherapy, nasogastric tube use, length of stay>14 days and intra-abdominal surgery, defined daily dose of antimicrobial agents≥19, prior use of more than three antimicrobial agents, and use of carbapenems were independent risk factors for the first episode of CDI. Use of laxatives, the first- and second-generation narrow-spectrum cephalosporins or metronidazole was identified as protective factors. It is necessary to make testing of C. difficile available as a routine practice and control these risk factors in Chinese hospitals to avoid CDI outbreaks.