Background: Zonulin has emerged as valuable biological marker to assess the integrity of the intestinal mucosal barrier. Recent evidence has shown that elevated serum zonulin levels are associated with increased intestinal permeability and has suggested a possible association between serum zonulin levels with obesity, dyslipidemia, insulin resistance and diabetes. However, the mechanisms underlying these associations are still unknown. Aim: To evaluate the relationship between serum zonulin concentration, fasting metabolic parameters, visceral (VAT) and subcutaneous adipose tissue (SAT), ectopic fats, oxidative stress markers and the composition of the habitual diet in individuals with type 2 diabetes (T2D). Methods: Correlation analyses were performed between fasting serum zonulin and fasting plasma glucose, insulin and lipids concentration, liver fat, pancreatic fat, VAT and SAT in 31 overweight/obese individuals (19M/12F) with type 2 diabetes, aged 35-70 years, participating in a nutritional intervention study (MEDEA study) with a diet rich in MUFA, PUFA, fibre, polyphenols, and vitamins or a MUFA rich diet. Correlations were assessed at baseline, before starting the dietary treatment. Serum zonulin was measured by ELISA. Liver, pancreatic fat, VAT and SAT were assessed by MRI. The composition of the participants’ habitual diet was evaluated by the 7-day food record. Results: Serum zonulin was 38% higher in men than women (40.1±5.9 ng/ml vs 29.1±7.9 ng/ml, p<0.001). Zonulin correlated directly with VAT (r=0.396, p=0.041) and urinary isoprostanes (r=0.434, p=0.017), but not with liver (r=-0.291; p=0.125), pancreatic fat (r=-0.245; p=0.085), SAT (r=-0.245; p=0.217), and other metabolic parameters. No correlation between serum zonulin and dietary components was observed. Conclusion: Our results showed a positive association between serum zonulin, VAT and urinary isoprostanes levels in subjects with T2D. Probably, a higher accumulation of visceral fat could upregulate zonulin expression and thus contribute to the dysfunction of the intestinal barrier and the worsening of oxidative stress.