To explore the effect of manno-oligosaccharide (MOS) on intestinal health in weaned pigs upon enterotoxigenic Escherichia coli K88 (ETEC) challenge, thirty-two male weaned pigs were randomly assigned into four groups. Pigs fed with a basal diet or basal diet containing MOS (0·6 g/kg) were orally infused with ETEC or culture medium. Results showed that MOS significantly elevated the digestibility of crude protein and gross energy in both ETEC-challenged and non-challenged pigs (P < 0·05). MOS also elevated serum concentrations of IgA and IgM (P < 0·05), but decreased serum concentrations of TNF-α, IL-1β and IL-6 (P < 0·05) in ETEC-challenged pigs. Interestingly, MOS increased villus height and the ratio of villus height:crypt depth in duodenum and ileum (P < 0·05). MOS also increased duodenal sucrase and ileal lactase activity in ETEC-challenged pigs (P < 0·05). MOS decreased the abundance of E. coli, but increased the abundance of Lactobacillus, Bifidobacterium and Bacillus in caecum (P < 0·05). Importantly, MOS not only elevated the expression levels of zonula occludens-1 (ZO-1), claudin-1 and GLUT-2 in duodenum (P < 0·05) but also elevated the expression levels of ZO-1, GLUT-2 and L-type amino acid transporter-1 in ileum (P < 0·05) upon ETEC challenge. These results suggested that MOS can alleviate inflammation and intestinal injury in weaned pigs upon ETEC challenge, which was associated with suppressed secretion of inflammatory cytokines and elevated serum Ig, as well as improved intestinal epithelium functions and microbiota.