Abstract Background Zonulin is involved in the integrity and functioning of both intestinal-epithelial barrier and blood–brain barrier (BBB) by regulating tight junction molecular assembly. Aim Since changes in microbiota and BBB may play a role in neurodegenerative disorders, we aimed to determine whether serum zonulin levels change in older patients affected by different types of dementia or mild cognitive impairment (MCI). Methods We evaluated serum zonulin levels in patients with late-onset AD (LOAD), vascular dementia (VAD), MIXED (AD + VAD) dementia, amnestic MCI, and in healthy controls. Results Compared with controls, serum zonulin increased in LOAD, MIXED dementia, and aMCI but not in VAD, independent of potential confounders (ANCOVA p = 0.01; LOAD vs controls, p = 0.01; MIXED vs. controls, p = 0.003; aMCI vs. controls, p = 0.04). Notably, aMCI converting to dementia showed significantly higher levels of zonulin compared with stable aMCI (p = 0.04). Serum zonulin inversely correlated with the standardized Mini-Mental State Examination (MMSE) score (p < 0.05), regardless of potential confounders. Discussion We found increased serum zonulin levels in patients with aMCI, LOAD and MIXED dementia, but not in VAD; moreover, zonulin levels were higher in aMCI converting to AD compared with stable ones. Conclusions Our findings suggest that a dysregulation of intestinal-epithelial barrier and/or BBB may be an early specific event in AD-related neurodegeneration.