Background: The mucus that lines the airway epithelium provides a barrier against pathogenic and noxious agents and participates in the innate mucosal response to inflammation and infection. Mucins are the major components of mucus and the macromolecules that impart rheologic properties to airway mucus. Airway mucus is overproduced in chronic rhinosinusitis (CRS). Biochemical and biophysical characterization of mucus in CRS and in normal airways will elucidate important aspects of CRS pathophysiology and allow the design of targeted medical treatments. The aim of this study was to estimate secretion of sinus mucus mucins in healthy individuals and CRS and correlate them with mucus biophysical properties. Methods: Twenty-seven sinus mucus samples from 21 patients were collected (14 subjects with CRS undergoing sinus surgery as part of their treatment and 7 control subjects undergoing hypophysectomy without sinonasal disease). Biophysical properties of the mucus were measured by rheometry. ELISA was done to estimate MUC5AC and MUC5B mucin content in comparison with standards, i.e., porcine gastric mucin (MUC5AC) and human salivary mucin (MUC5B). Results: MUC5B secretion ± SEM was 0.49 ± 0.16 μg/mL (n = 14) and 0.17 ± 0.05 μg/mL (n = 7) and MUC5AC secretion ± SEM was 1.26 ± 0.26 μg/mL (n = 14) and 1.46 ± 0.61 μg/mL (n = 7) in chronic sinusitis and control subjects, respectively. There was linear correlation between viscosity and mucin content in the control group but not in the CRS group. Conclusion: MUC5B secretion is significantly up-regulated in CRS compared with control subjects (p = 0.04). Correlation between viscosity and mucin content was lost in CRS. This is likely to have important implications for future therapies in CRS.