To study the pleiotropic effects of vitamin D in airway host defense, we use airway epithelial organoids cultured on air-liquid interface (ALI), originating from three chronic rhinosinusitis patients, marking as N66, N67 and N69, respectively. 24 hours prior to infection of Staphylococcus Aureus or influenza virus H1N1, 10nM and 100nM 1,25(OH)2D3 were added respectively to the medium in the lower chamber of Transwells with organoids. 24 hours post infection, 100μl DPBS were added to the apical surface of organoids for 20 minutes and then cellular secretion diluted in the DPBS were collected for proteomics analysis. The control groups without 1,25(OH)2D3 treatment were denoted as ND and those without infection were denoted as C. We applied a mass spectrometry-based tandem mass tag quantitative proteomics to analyze the cellular secretion from airway epithelial organoids, so as to study the host defense effects of vitamin D against pathogen infection.