Background: Povidone-iodine (PVP-I) is an antiseptic and a disinfectant with broad-spectrum antimicrobial activity against various pathogens. However, it is unclear whether PVP-I nasal instillation can suppress mucosal inflammation in non-eosinophilic chronic rhinosinusitis (CRS) mice. Objective: This study aimed to explore the anti-inflammatory effects and underlying molecular mechanism of PVP-I on lipopolysaccharide-stimulated airway epithelial cells and investigate whether nasal instillation of PVP-I can suppress mucosal inflammation in non-eosinophilic CRS mice. Methods: Inflammation-related molecules in the nasal epithelial cells and non-eosinophilic CRS mice were measured by enzyme-linked immunosorbent assay, western blotting, quantitative real-time polymerase chain reaction, immunoprecipitation, and histopathological analysis (hematoxylin and eosin staining, immunohistochemistry, and periodic acid‑schiff staining). Results: PVP-I blocked expressions of various inflammation-related molecules, such as NLRP3, NF-κB-p65, caspase-1, and IL-1β; induced translocation of NF-κB to the nucleus; and promoted assembly of NLRP3/ASC complexes in the nasal epithelial cells and non-eosinophilic CRS mice. Notably, PVP-I strongly blocked the receptor interaction of TLR4 and MyD88 in the epithelial cells of nasal mucosa. Conclusion: We demonstrated that PVP-I significantly attenuated inflammatory molecules and cytokines via blocking the formation of TLR4 and MyD88 complexes during LPS-induced mucosal inflammation in non-eosinophilic CRS.