Aluminum (Al³⁺) is a widespread environmental metal with potential effects on the intestinal epithelium. In this study, we evaluated the impact of dietary concentrations of aluminum on barrier integrity, histological organization, and gene expression in a 3D human intestinal tissue model. Tissues were exposed to increasing concentrations of aluminum (0, 5, 20, and 50 ppm) for 12 days to mimic environmental chronic exposure through ingestion. Transepithelial electrical resistance (TEER) measurements revealed a significant decrease only at the highest dose (50 ppm) on day 12, indicating a damage to the epithelial permeability. This was supported by a marked downregulation of OCLN and CLDN-5 expression, key genes involved in tight junction integrity. Aluminum permeation across the tissue was confirmed via Inductively Coupled Plasma—Atomic Emission Spectroscopy (ICP-AES), indicating dose-dependent penetration. Furthermore, gene expression analysis showed significant upregulation of MT-1E and MT-2A (markers of cellular response to metal exposure) while MT-1A remained unchanged. Histological analysis showed a vacuolated aspect in the tissue treated with 50 ppm. Confocal imaging revealed an apical accumulation of F-actin at the highest concentration of Al ³⁺ and transmission electron microscopy (TEM) demonstrated progressive shortening of apical microvilli in aluminum-treated tissues. The present data demonstrate that repeated exposure to 50 ppm of Al ³⁺ impacts intestinal barrier function and alters the tissue organization in the human intestinal epithelium.