The functional differences among individual prebiotics are largely unknown, and different prebiotics are usually used as functional composition analogues for food addition and clinical intervention. To investigate the functional specificity of 6 widely-used prebiotics (GF2,2′-FL, FOS, GOS, MOS and inulin) on inflammation and immunity, which could provides guidance and experimental evidence for precise nutrition, a model of intestinal epithelial cell inflammation was firstly constructed by using EHEC (enterohemorrhagic Escherichia coli O157:H7) to stimulate HCT-8 cells. And the effects of six prebiotics on cell inflammation were analyzed via detecting the activation of NF-κB signaling pathway and expression levels of proteins TNF-α and IL-6 in its downstream. Then a in vitro dentritic cell maturation assay was constructed. Human dentritic cells were obtained by density gradient centrifugation, and used GM-CSF and IL-4 cytokines to stimulate PBMC (peripheral blood mononuclear cells) separated from 30 volunteers and treated with or without prebiotics. The effects of six prebiotics treatment on dentritic cells maturation were determined by comparing the proportion of mature dentritic cells through flow cytometry. The results showed that only 2′-FL, GOS and GF2 treatment could inhibit the EHEC-induced HCT-8 cell inflammation. In addition, 2′-FL, GF2 and inulin treatment promoted dentritic cells maturation (increase rate 73.3%, 57.1% and 63.3%, respectively), GOS treatment significantly inhibited dentritic cells maturation (decrease rate 73.3%). Therefore, these findings suggested that the effects on regulating immunity varied among different prebiotics and prebiotics addition required precise design according to the physical condition of individuals.