Aichi virus has been described as a novel causative agent of gastroenteritis in humans. In this study, we report the seroprevalence distribution of Aichi virus in Tunisia. A panel of 1,000 sera was screened by applying an enzyme-linked immunosorbent assay for immunoglobulin G specific for Aichi virus. A considerable prevalence (92%) of antibody to Aichi virus was found across all age groups. The specific anti-Aichi virus antibodies increased with age, from a high rate (68.8%) in children under 10 years old to about 100% in persons more than 60 years old. We found a statistically significant increase in levels of antibody to Aichi virus according to the age of patients. Immunoglobulin M antibodies were detected among five children. A high frequency of Aichi virus monoinfections in hospitalized children with severe gastroenteritis was previously observed in Tunisia. Aichi virus causes diarrhea with dehydration, fever, and vomiting. This work is the first to establish a correlation between the high seroprevalence of specific Aichi virus antibodies, clinical presentation, and a high frequency of isolation of Aichi virus by genomic characterization in stools of children suffering from gastroenteritis. Our data show the importance and emerging character of Aichi virus in the viral etiology of pediatric gastroenteritis.