Background: Clostridium difficile infection (CDI) has an increasing pediatric prevalence worldwide. However, molecular characteristics of C. difficile in Chinese children with acute gastroenteritis have not been reported. Methods: A five-year cross-sectional study was conducted in a tertiary children's hospital in Zhejiang. Consecutive stool specimens from outpatient children with acute gastroenteritis were cultured for C. difficile, and isolates then were analyzed for toxin genes, multi-locus sequence type and antimicrobial resistance. Diarrhea-related viruses were detected, and demographic data were collected. Results: A total of 115 CDI cases (14.3%), and 69 co-infected cases with both viruses and toxigenic C. difficile, were found in the 804 stool samples. The 186 C. difficile isolates included 6 of toxin A-positive/toxin B-positive/binary toxin-positive (A + B + CDT +), 139 of A + B + CDT − , 3 of A − B + CDT + , 36 of A − B + CDT − and 2 of A − B − CDT −. Sequence types 26 (17.7%), 35 (11.3%), 39 (12.4%), 54 (16.7%), and 152 (11.3%) were major genotypes with significant differences among different antimicrobial resistances (Fisher's exact test, P < 0.001). The A − B + isolates had significantly higher resistance, compared to erythromycin, rifampin, moxifloxacin, and gatifloxacin, than that of the A + B + (χ 2 = 7.78 to 29.26, P < 0.01). The positive CDI rate in infants (16.2%) was significantly higher than that of children over 1 year old (10.8%) (χ 2 = 4.39, P = 0.036). Conclusions: CDI has been revealed as a major cause of acute gastroenteritis in children with various genotypes. The role of toxigenic C. difficile and risk factors of CDI should be emphatically considered in subsequent diarrhea surveillance in children from China.