Human milk oligosaccharides (HMO), complex sugars that are highly abundant in breast milk, block viral and bacterial attachment to the infant's intestinal epithelium and lower the risk of infections. We hypothesised that HMO also prevent infections with the protozoan parasite Entamoeba histolytica, as its major virulence factor is a lectin that facilitates parasite attachment and cytotoxicity and binds galactose (Gal) and N-acetyl-galactosamine. HMO contain Gal, are only minimally digested in the small intestine and reach the colon, the site of E. histolytica infection. The objective of the present study was to investigate whether HMO reduce E. histolytica attachment and cytotoxicity. Our in vitro results show that physiological concentrations of isolated, pooled HMO detach E. histolytica by more than 80%. In addition, HMO rescue E. histolytica-induced destruction of human intestinal epithelial HT-29 cells in a dose-dependent manner. The cytoprotective effects were structure-specific. Lacto-N-tetraose with its terminal Gal rescued up to 80% of the HT-29 cells, while HMO with fucose α1-2-linked to the terminal Gal had no effect. Galacto-oligosaccharides (GOS), which also contain terminal Gal and are currently added to infant formula to mimic some of the beneficial effects of HMO, completely abolished E. histolytica attachment and cytotoxicity at 8mg/ml. Although our results need to be confirmed in vivo, they may provide one explanation for why breast-fed infants are at lower risk of E. histolytica infections. HMO and GOS are heat tolerant, stable, safe and in the case of GOS, inexpensive, which could make them valuable candidates as alternative preventive and therapeutic anti-amoebic agents. © 2012 The Authors.