Autism spectrum disorder (ASD) is a common neurodevelopmental disorder characterized by limitations in mutual communication and social interaction as well as restricted, repetitive patterns of behaviors, interests, or activities. The possible role of biological abnormalities in the etiopathogenesis of this disorder arouses research interest in this area. This is a case-control study evaluating epithelial barrier function by comparing serum concentrations of occludin and zonulin in children with ASD (n = 60) and controls (n = 30). The Childhood Autism Rating Scale (CARS) was used to evaluate autism symptom levels in all children. Serum occludin and zonulin levels were analyzed using an enzyme-linked immunosorbent assay. Serum occludin was significantly lower in children with ASD than in control subjects. In children with ASD, a decrease in occludin level was significantly associated with the disorder symptom levels items mean score (CARS total scores). Our findings showed that children with ASD had alterations in epithelial barrier function compared to the control group. The investigation of the mechanism underlying the different levels of occludin between ASD and controls may be of importance in clarifying the etiopathogenesis of ASD, as well as its follow-up and treatment.