Background: Microbial components in the environment are potent activators of the immune system with capacity to shift the active immune response towards priming of Th1 and/or Th2 cells. Lipopolysaccharide (LPS), a cell-wall component of Gram-negative bacteria, is extensively present in food products like cow's milk. It is not well established, however, how this presence of LPS affects oral tolerance induction. Methods: We studied the effect of LPS contamination in a commercial preparation of the cow milk protein β-lactoglobulin (β-LG) on antigen-specific immune responses. IgG1/IgG2a production upon intraperitoneal immunization without adjuvant was measured, and oral tolerance induction against β-LG after administration of either an aqueous solution or water-in-oil (w/o) emulsion of β-LG was evaluated. Results: LPS contamination of β-LG provoked a β-LG-specific IgG2a response, as well as an enhanced β-LG-specific IgG1 response upon intraperitoneal immunization. Oral tolerance induction to β-LG was induced by aqueous solutions of β-LG with and without LPS administration. Conversely, oral administration of w/o-emulsified β-LG prevented oral tolerance to β-LG only when the β-LG was contaminated with LPS. Conclusions: LPS contamination of an aqueous protein solution does not affect oral tolerance induction, whereas LPS present in emulsion prevents oral tolerance induction towards the food protein.