Background: The trigger of the autoimmune destruction of pancreatic beta cells in Type 1 diabetes (T1D) is unclear. One theory is that antigens absorbed through the gut may be involved. We have recently described a protein, zonulin, that opens intestinal tight junctions and allows paracellular absorption of macromolecules. We have reported that zonulin is increased in the serum of a subset of patients with T1D and first degree relatives. Aim: The objective of this study was to determine if dysregulation of the zonulin pathway, both at baseline and following a gluten-containing meal, is linked to increased intestinal permeability (IP) and is involved in the pathogenesis of T1D. Methods: After obtaining informed consent, blood was obtained from children with T1D, parents, and siblings. Zonulin was measured in the serum of children with T1D (n=11) and their first degree relatives (n=19), by sandwich ELISA. IP was determined by HPLC measurement of both serum (at baseline and every hour up to 5 hours) and urine (5 hours collection) lactulose (LA) and mannitol (MA) after subjects ingested the sugars test solution. Results were expressed as LA/MA ratio. Results: There was a significant increase in zonulin (53%) and IP (47 %) in subjects compared to the negative cutoff. There was a significant relationship between elevation of zonulin and increased IP (p=0.026) and between LA/MA ratio in post-prandial serum samples and LA/MA ratio in urine (p=0.01). Although there was no change in mean zonulin levels with a meal, all subjects for whom IP data is available had elevation of zonulin at baseline and post-meal, and all had an increase in IP 1-hour post-meal. The 1 h post-prandial IP increase was statistically higher in T1D subjects (5.6 folds) as compared to their relatives (2.7 folds, p<0.01). There was no relationship between serum glucose and zonulin levels. Conclusions: Zonulin is significantly correlated with increased IP in children with T1D and their first degree relatives. These data suggest that increased serum zonulin is indicative of increased gut leakiness in children with T1D. Studies of the temporal relationship of an increase in serum zonulin and the development of diabetes autoantibodies are planned to suggest a causative relationship in a subset of genetically susceptible individuals.