Low serum levels of 1α, 25-dihydroxyvitamin D3 (VD3) are associated with a higher mortality in trauma patients with sepsis or ARDS. However, the molecular mechanisms behind this observation are not yet understood. VDis known to stimulate lung maturity, alveolar type II cell differentiation, or pulmonary surfactant synthesis and guides epithelial defense during infection. In this study, we investigated the impact of VDon the alveolar-capillary barrier in a co-culture model of alveolar epithelial cells and microvascular endothelial cells respectively in the individual cell types. After stimulation with bacterial LPS (lipopolysaccharide), gene expression of inflammatory cytokines, surfactant proteins, transport proteins, antimicrobial peptide, andwere analyzed by real-time PCR, while corresponding proteins were evaluated by ELISA, immune-fluorescence, or Western blot. The effect of VD3 on the intracellular protein composition in H441 cells was analyzed by quantitative liquid chromatography-mass spectrometry-based proteomics. VDeffectively protected the alveolar-capillary barrier against LPS treatment, as indicated by TEER measurement and morphological assessment. VDdid not inhibit the IL-6 secretion by H441 and OEC but restricted the diffusion of IL-6 to the epithelial compartment. Further, VDcould significantly suppress the surfactant protein A expression induced in the co-culture system by LPS treatment. VDinduced high levels of the antimicrobial peptide LL-37, which counteracted effects by LPS and strengthened the barrier. Quantitative proteomics identified VD3-dependent protein abundance changes ranging from constitutional extracellular matrix components and surfactant-associated proteins to immune-regulatory molecules. DCLK1, as a newly described target molecule for VD, was prominently stimulated by VD(10 nM) and seems to influence the alveolar-epithelial cell barrier and regeneration.