Drug delivery systems (DDS) for oral delivery of peptide drugs contain excipients that facilitate and enhance absorption. However, little knowledge exists on how DDS excipients such as permeation enhancers interact with the gastrointestinal mucus barrier. This study aimed to investigate interactions of the permeation enhancer sodium 8-[(2-hydroxybenzoyl)amino]octanoate (SNAC) with ex vivo porcine intestinal mucus (PIM), ex vivo porcine gastric mucus (PGM), as well as with in vitro biosimilar mucus (BM) by profiling their physical and barrier properties upon exposure to SNAC. Bulk mucus permeability studies using the peptides cyclosporine A and vancomycin, ovalbumin as a model protein, as well as fluorescein-isothiocyanate dextrans (FDs) of different molecular weights and different surface charges were conducted in parallel to mucus retention force studies using a texture analyzer, rheological studies, cryo-scanning electron microscopy (cryo-SEM), and single particle tracking of fluorescence-labelled nanoparticles to investigate the effects of the SNAC-mucus interaction. The exposure of SNAC to PIM increased the mucus retention force, storage modulus, viscosity, increased nanoparticle confinement within PIM as well as decreased the permeation of cyclosporine A and ovalbumin through PIM. Surprisingly, the viscosity of PGM and the permeation of cyclosporine A and ovalbumin through PGM was unaffected by the presence of SNAC, thus the effect of SNAC depended on the regional site that mucus was collected from. In the absence of SNAC, the permeation of different molecular weight and differently charged FDs through PIM was comparable to that through BM. However, while bulk permeation of neither of the FDs through PIM was affected by SNAC, the presence of SNAC decreased the permeation of FD4 and increased the permeation of FD150 kDa through BM. Additionally, and in contrast to observations in PIM, nanoparticle confinement within BM remained unaffected by the presence of SNAC. In conclusion, the present study showed that SNAC altered the physical and barrier properties of PIM, but not of PGM. The effects of SNAC in PIM were not observed in the BM in vitro model. Altogether, the study highlights the need for further understanding how permeation enhancers influence the mucus barrier and illustrates that the selected mucus model for such studies should be chosen with care.