Cyclic peptides are promising prospects for orally administered medicines, but limited intestinal permeability has hampered their development. Although certain biological mechanisms that affect cyclic peptide intestinal permeability have been identified, the impact of the mucus barrier, a key stumbling block to epithelial medication delivery, on cyclic peptide bioavailability remains unknown. We show that the lipophilic cyclic peptide cyclosporin A (CsA) interacted with polymeric, gel-forming mucins (MUC2, MUC5AC, and MUC5B), which support the mucus gel-networks in the gastrointestinal tract, and likely caused aggregation. Two additional cyclic peptides (daptomycin and polymyxin B) did not produce mucin aggregation under the same circumstances. Purified MUC2, MUC5AC, and MUC5B mucins sedimented quicker in the presence of CsA using rate-zonal centrifugation, with a substantial increase in mucins in the pellet fraction. Mucin sedimentation patterns, on the other hand, were substantially unaffected by daptomycin or polymyxin B therapy. CsA promoted MUC5B sedimentation in a concentration-dependent manner, and sedimentation tests using recombinant mucin protein domains indicate that CsA is most likely responsible for aggregation of MUC5B's largely non-O-glycosylated N-terminal and C-terminal regions. Furthermore, pH influenced the aggregation of the N-terminal area but not the C-terminal region.