Peptidoglycan recognition protein 3 (PGlyRP3) is a pattern recognition protein found in the gut epithelia and proven to have an anti-inflammatory effect in response to dietary lipids via the upregulation of PPARγ. We have reported an in vitro anti-inflammatory action for prebiotic oligosaccharides in the intestinal Caco-2 cells through stimulation of PPARγ and PGlyRP3. In the present study, this effect was examined in vivo in a DSS-induced colitis mouse model. Both Raftilose P95 and α3-Sialyllactose oligosaccharides upregulated PGlyRP3 and showed an anti-colitis effect as evidenced by increased survival and reductions in body weight loss, bleeding in stool, diarrhea, and the expression of the cytokine genes IL6, IL1b and TNFα. However, unlike the in vitro results, the role of PPARγ was unclear; both oligosaccharides could upregulate neither PPARγ gene nor protein expressions, nor the administration of its inhibitor BADGE could affect the upregulation of PGlyRP3 in response to oligosaccharides. Instead, phosphoimmunoblotting indicated that both oligosaccharides inhibit the phosphorylation of MEK1/2 and ERK1/2 caused by DSS. Also, both oligosaccharides reversed the DSS-induced downregulation in the gene expression of IκBα, the upregulation of NFκB1 and cox2, and the decrease of the cytoplasmic (inactive) NF-κB p65 protein that indicates its transformation to the (active) inflammatory nuclear form. The present study showed that the prebiotic oligosaccharides Raftilose P95 and α3-Sialyllactose have an anti-colitis effect in mice through the induction of PGlyRP3 expression and down-regulation of the MEK/ERK and the NF-κB inflammatory pathways.Copyright © 2020 Elsevier Inc. All rights reserved.