Leaky gut hypothesis is one of the well-known theory which tries to explain etiology of Autism Spectrum Disorder (ASD). Unfortunately there is still a gap of evidence to investigate the corner points of the hypothesis. The aim of this study was to investigate the determinants of leaky gut in children with ASD, their siblings and healthy controls. Intestinal microbiota was found to be similar between ASD and sibling groups. Biological markers of bacterial translocation showed a significant difference in the sibling group, whereas the marker indicating local inflammation was not different between the groups. The findings from this study did not support the role of Gut microbiota or leaky gut on the etiology of autism.