Background: Tight junctions (TJs) are intracellular structures which are essential for epithelial barrier function and play an important role in antimicrobial defense. Epithelium dysfunction and type-2-skewed inflammation are two main pathological phenomena of chronic rhinosinusitis with nasal polyps (CRSwNP). However, the effect of pro-inflammatory type-2 cytokine interleukin-13 (IL-13) on TJs in CRSwNP is poorly understood. Methods: Nasal biopsies of 70 CRSwNP patients and 25 healthy subjects, and in vitro IL-13-matured human nasal epithelial cells (hNECs) in 9 persons were used to analyze epithelial markers and TJ proteins. Epithelium permeability, transepithelial electrical resistance (TEER), mRNA and protein expression of TJs were quantified for IL-13-matured hNECs and that with RV infection. Results: Both mRNA and protein expression of occludin, claudin-3 and ZO-1 were significantly decreased in CRSwNP biopsies and in hNECs after IL-13 treatment. Differentiation of hNECs with IL-13 treatment increased epithelium permeability, decreased TEER and altered hNECs composition resulting in lesser ciliated cells and mucus over-secretion. Interestingly, claudin-3 is selectively expressed on ciliated cells. While RV infection induced minimal changes to TJs, the IL-13-matured hNECs has reduced capacity for upregulation of IFN-λ1 and CXCL10 but further increased the expression of TSLP upon acute RV infection. Conclusions: These findings suggested that IL-13-mediated dysfunction of TJs and compromised epithelial barrier. IL-13-induced cilia loss conferred lowered viral replication and impaired antiviral responses of nasal epithelium against acute RV infection.