Clostridium difficile infection is almost unrecognized in mainland China. We have undertaken a study in a large Chinese teaching hospital in Changsha, Hunan, China, to identify cases of C. difficile, record patient characteristics, and define the molecular epidemiology with respect to ribotype distribution and cross-infection. Between April 2009 and February 2010, we examined fecal samples from 70 hospitalized patients with diarrhea who were receiving or had received antibiotics within the previous 6 weeks. Clinical information was collected and the samples were cultured for C. difficile retrospectively. Isolates were ribotyped, and multiple-locus variable-number tandem-repeat assay (MLVA) subtyping was performed on clusters of the same ribotype. The mean age of patients from whom C. difficile was cultured was 58 years, with only 4/21 patients aged > 65 years. All patients, with a single exception, had received a third-generation cephalosporin and/or a quinolone antibiotic. Twenty-one isolates of C. difficile were recovered, and seven different ribotypes were identified, the dominant types being 017 (48%), 046 (14%), and 012 (14%). We identified two clusters of cross-infection with indistinguishable isolates of ribotype 017, with evidence of spread both within and between wards. We have identified C. difficile as a possibly significant problem, with cross-infection and a distinct ribotype distribution, in a large Chinese hospital. C. difficile may be underrecognized in China, and further epidemiological studies across the country together with the introduction of routine diagnostic testing are needed to ascertain the size of this potentially significant problem.