Background Mucus overlies the nasal mucosa in the respiratory and olfactory regions, which perform distinct physiological functions. However, proteomic differences between respiratory mucosa (RM) and olfactory cleft (OC) mucus remain unclear.Methods Mucus samples were collected from the RM and OCs of 25 healthy participants (mean age: 24.8 years [SD 4.16]; 12 women, 13 men) using cotton and viscose strips. Proteomic profiling was performed using nano liquid chromatography-electrospray ionization tandem mass spectrometry (nanoLC-MS/MS), and enriched proteins were analyzed for functional associations. Olfactory testing was performed using the Sniffin' Sticks odor identification test.Results RM mucus contained a greater number of enriched proteins, including complement components (e.g., C3, C4), neutrophil antimicrobial proteins (e.g., myeloperoxidase, neutrophil elastase, S100 proteins), and coagulation-related/proteolytic proteins (e.g., fibrinogen, plasminogen, vitronectin, alpha-2-macroglobulin). These proteins were associated with immune defense, coagulation, and regulatory pathways. OC mucus contained fewer enriched proteins, including aldehyde dehydrogenase family 3 member B1, chloride intracellular channel protein 6, long-chain fatty acid transport protein 2, and stromelysin-2, supporting epithelial maintenance, ionic homeostasis, and odorant metabolism. Overrepresented processes in OC mucus were linked to ciliary structure/motility and epithelial maintenance.Conclusions Proteomic profiles reflect regional specialization: RM mucus is optimized for immune response, whereas OC mucus maintains a microenvironment conducive to sensory function and epithelial integrity. Collectively, the results highlight how regional mucus proteomic differences parallel the distinct physiological roles of the respiratory and olfactory epithelia and provide a foundation for investigating proteomic changes in olfactory dysfunction or nasal inflammatory disease.