Human milk oligosaccharides(HMOs)， a key active ingredient in human milk， have important physiological functions such as regulating intestinal microbiota， protecting intestinal barrier integrity， and regulating immunity. This study constructed the Caco-2 intestinal epithelial barrier dysfunction model by the treatment of the pro-inflammatory cytokines mixture. Three HMOs including neutral fucosylated， sialylated and neutral non-fucosylated HMOs， namely 2'-fucosyllactose (2'-FL)， 3'-sialyllactose (3'-SL) and lacto-N-neotetraose (LNnT)， were used for treatment. In order to explore the protective effect of HMOs on intestinal barrier function and the regulatory mechanism， this study characterized the intestinal barrier integrity by measuring transepithelial electrical resistance (TEER)， determined the secretion of inflammatory factors by enzyme-linked immunosorbent assay， and analyzed the expression level of tight junction and inflammation related genes by quantitative real-time PCR. The results showed that all three HMOs could increase TEER in a concentration dependent manner. All HMO treatments could reduce the secretion of IL-6 and IL-8， inhibit the gene expression level of IL-6， IL-8， IL-1β， TNF-α， COX-2 and iNOS， enhance the transcriptional activity of Claudin-1， Claudin-3， ZO-1， ZO-2， and Occludin， and inhibit the activation of NF-κB signaling pathway without concentration dependence. All HMO treatments could exert significant intestinal barrier protection and anti-inflammatory activity at high mass concentrations (2.4 mg/mL and 10 mg/mL). Our results provided scientific evidence for the HMO application to enhance intestinal barrier function and immunomodulatory function.