Lipopolysaccharide was found to be elevated in the plasma of necrotizing enterocolitis (NEC) and inflammatory bowel disease (IBD) patients and may play an important role in the pathogenesis and propagation of these intestinal diseases. To illustrate the destructive effect of lipopolysaccharide (LPS) and to test the protective effect of 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) on LPS-induced barrier injury, an in vitro intestinal epithelia barrier model was established with Caco-2 monolayers and treated with clinically relevant concentrations (1-10 ng/ml) of LPS with or without 1,25(OH)2D3. Transepithelial electrical resistance (TEER) and FITC-Dextran 40kda (FD-40) flux were measured to reflect monolayer permeability. We found that LPS at clinically relevant concentrations increased intestinal permeability by downregulating and redistributing tight junction (TJ) proteins. 1,25(OH)2D3 added at baseline or at day 4 abrogated the destructive effect of LPS on monolayer permeability by restoring the expression and localization of TJ proteins. LPS, at clinically relevant concentrations, also downregulated the expression of vitamin D receptor (VDR); 1,25 (OH)2D3, however, could restore the expression of VDR. Our findings illustrate the mechanism underlying the destructive effect of clinically relevant concentrations of LPS on intestinal TJ barrier and provide evidence for the clinical application of vitamin D in LPS-related intestinal barrier dysfunction.