We recently showed that adherence to the Mediterranean diet increased the proportion of plasma omega-3 polyunsaturated fatty acids (n-3 PUFAs), which was associated with an improved intestinal barrier integrity. In the present exploratory analysis, we assessed fecal fatty acids in the same cohort, aiming to investigate possible associations with intestinal barrier integrity. Women from the LIBRE randomized controlled trial, characterized by an impaired intestinal barrier integrity, followed either a Mediterranean diet (intervention group, IG, n=33) or a standard diet (control group, CG, n=35). At baseline (BL), month 3 (V1), and month 12 (V2), plasma lipopolysaccharide binding protein (LBP), fecal zonulin, and fecal fatty acids were measured. In the IG, fecal proportions of palmitoleic acid (16:1, n-7) and arachidonic acid (20:4, n-6) decreased, while the proportion of linoleic acid (18:2, n-6) and alpha linoleic acid (18:3, n-3) increased (BL-V1 and BL-V2, all P<0.08). In the CG, fecal proportions of palmitic acid and arachidic acid increased while the proportion of linoleic acid decreased (BL-V1, all P<0.05). The decrease in the proportion of palmitoleic acid correlated with the decrease in plasma LBP (∆V1-BL r=0.72, P<0.001; ∆V2-BL r=0.39, P<0.05) and correlated inversely with adherence to the Mediterranean diet (Mediterranean diet score; ∆V1-BL r=-0.42, P=0.03; ∆V2-BL r=-0.53, P=0.005) in the IG. Our data show that adherence to the Mediterranean diet induces distinct changes in the fecal fatty acid composition. Furthermore, our data indicate that the fecal proportion of palmitoleic acid, but not fecal n-3 PUFAs, are associated with intestinal barrier integrity in the IG.