Cashew nuts are rich in dietary fibers, monounsaturated fatty acids, carotenoids, tocopherols, flavonoids, catechins, amino acids, and minerals that offer benefits for health. However, the knowledge of its effect on gut health is lacking. In this way, cashew nut soluble extract (CNSE) was assessed in vivo via intra-amniotic administration in intestinal brush border membrane (BBM) morphology, functionality, and gut microbiota. Four groups were evaluated: (1) no injection (control); (2) H2O injection (control); (3) 10 mg/mL CNSE (1%); and (4) 50 mg/mL CNSE (5%). Results related to CNSE on duodenal morphological parameters showed higher Paneth cell numbers, goblet cell (GC) diameter in crypt and villi, depth crypt, mixed GC per villi, and villi surface area. Further, it decreased GC number and acid and neutral GC. In the gut microbiota, treatment with CNSE showed a lower abundance ofBifidobacterium,Lactobacillus, andE. coli. Further, in intestinal functionality, CNSE upregulated aminopeptidase (AP) gene expression at 5% compared to 1% CNSE. In conclusion, CNSE had beneficial effects on gut health by improving duodenal BBM functionality, as it upregulated AP gene expression, and by modifying morphological parameters ameliorating digestive and absorptive capacity. For intestinal microbiota, higher concentrations of CNSE or long-term intervention may be necessary.