The intestinal compartment confines the gut microbiome while enabling food passage and absorption of active molecules. For the rational design of oral formulations aiming to overcome physiological barriers of the gut, it is crucial to understand how cells respond to the presence of nanoparticulate materials. Taking advantage of the versatility and biocompatibility of dendritic mesoporous silica nanoparticles (DMSNs), several post-grafting strategies are developed to diversify the surface properties of spherical DMSNs and then probe interactions with the intestinal coculture cell model Caco-2/HT29-MTX-E12. Herein, the functionalization of DMSNs with polyethylene glycol, phosphonate, methyl, and farnesol moieties enables the investigation of both particle penetration through the mucus layer and pathways relevant to intracellular uptake. Contributions of surface chemistry, charge, and colloidal stability are correlated with the modulation of particle movement through the mucus and the organization of cell-cell junctions. Hydrophilic and negative functionalities favor particle distribution toward the intestinal monolayer. Instead, hydrophobic DMSNs are hindered by the mucus, possibly limiting cell contact. Hybrid surfaces, combining phosphonate and long carbon chain functions, support diffusion through the mucus and foster the paracellular permeability as well as the transient barrier relapse, as indicated by increased cell-cell distances and reorganization of tight junctions.