Mannan from yeast has been demonstrated to limit infection in animals susceptible to gastrointestinal infection, including pigs, poultry and cows, by blocking the mechanism by which gram-negative bacteria adhere to and invade the intestines. Enterotoxigenic Escherichia coli (ETEC) cause post weaning diarrhoea (PWD) which results in poor weight gain and potential death at great economic cost to the farmer. A mannan rich fraction (MRF) was assessed in vitro for its impact on ETEC infection of HT-29 intestinal cell line. Gene expression markers for inflammation (TNFα and IL-1β) and TLR4 (TICAM-1 and LY96) associated recognition of bacteria were significantly elevated following exposure to E. coli alone, but not in combination with MRF compared to the control. HT-29 cells exposed to MRF alone demonstrated significantly reduced expression of immune signalling genes IRAK1, IRF7 and JUN when compared to the control. HT-29 cell protein abundance for TNFα and TLR4 associated proteins were significantly increased in response to E. coli exposure alone while no significant change was observed for MRF treatment with E. coli infection. E. coli adhesion to HT-29 cells was significantly decreased with addition of MRF compared to E. coli infection alone. The action of MRF demonstrated its potential capacity to limit infection on an in vitro level through blocking bacterial interaction with the intestines that leads to infection as marked by a reduction in proinflammatory responses. MRF on its own demonstrated potential anti-inflammatory effects on intestinal cells with the reduction of proinflammatory responses observed.