Zonulin is a protein associated with the tight junction complex opening at the intestinal epithelium, previously linked to obesity, cardiovascular diseases, type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD). However, its role in CKD has not been totally elucidated. This study aimed to evaluate zonulin levels in subjects with diabetic kidney disease (DKD). This case-control study included two cases groups: 1) Advanced DKD cases: T2DM patients with estimated glomerular filtration rate (eGFR) <60ml/min/1.73m2; 2) Albuminuric T2DM cases: diabetic patients with urinary albumin excretion (UAE) >30mg/g creatinine, but with eGFR>60ml/min/1.73m2. Two control groups were also included: 1) T2DM controls: patients with T2DM without impaired kidney function; 2) Non-T2DM controls: subjects without T2DM and normal renal function. Serum levels of zonulin were measured by ELISA. Eighty-six individuals were included. Zonulin levels was different among study groups (P = 0.003). T2DM controls presented higher zonulin levels than non-T2DM controls [(131.35 (83.0-170.5) vs. 87.25 (54.7-111.8), P = 0.018] and advanced DKD cases [63.72 (45.03-106.0); P = 0.007]. Zonulin showed a positive correlation with eGFR (r = 0.222; P = 0.040), total cholesterol (r = 0.299; P = 0.034), LDL (r = 0.258; P = 0.021), and negative with albuminuria (r = -0.243; P = 0.024) and body fat (r = -0.271; P = 0.014). In the multivariate logistic regression analyses, zonulin levels were independently associated to renal outcomes [OR 0.99 (0.98-0.99, P = 0.012)] after 5-year inclusion. In conclusion, increased zonulin levels in patients with TD2M without renal disease suggest an impaired intestinal permeability. Moreover, its association with renal outcomes could indicate its use as a disease monitoring marker. However, the mechanisms behind this association should be better understood.