Human parechoviruses (HPeVs) are widespread pathogens causing a wide spectrum of diseases. HPeVs belong to the family Picornaviridae, and 14 genotypes are known. We conducted a case-control study to investigate the role of HPeV in acute gastroenteritis. HPeV was detected and quantified using real-time RT-PCR, and then genotyped by sequencing of the nested RT-PCR product of the VP3/VP1 partial gene. HPeV was found in both the case and control groups (29.4% and 15.3% respectively, p 0.006). Six HPeV genotypes (HPeV1, HPeV3, HPeV4, HPeV5, HPeV6, and HPeV8) were detected. Nine positive samples could not be sequenced with negative genotyped RT-PCR. HPeV1 and HPeV3 were the most prevalent genotypes, and co-infection was common in the case group. No statistically significant differences in either viral load or the rate of HPeV1 and HPeV3 infection were found between the two groups. Additionally, no significant differences were found in fever rates, vomiting rates or mean duration and frequency of diarrhoea and vomiting between the positive and negative case groups with HPeV1 or HPeV3. Multivariate logistic regression analysis indicated that there was no association between the HPeV1 or HPeV3 infection and acute gastroenteritis. Multiple genotypes of HPeVs were highly prevalent in Chinese children. One potential new HPeV genotype was identified, but needs to be confirmed further by the picoma study group. However, the present study does not support a causative role of HPeV1 and HPeV3 in acute gastroenteritis.