<b><i>Background:</i></b> Little is known about the contribution of the invariant natural killer T (iNKT) cells in the onset of food allergy. Using a mouse model for cow’s milk allergy the function of iNKT cells was investigated. <b><i>Methods:</i></b> Mice were sensitized orally with casein or whey proteins. One hour before the sensitizations the mice were injected intraperitoneally with α-galactosylceramide (αGalCer) or control. One week after the last sensitization acute allergic skin reactions were measured. Furthermore, in the liver, spleen and mesenteric lymph nodes (MLN) percentages of iNKT cells were analyzed and liver lymphocyte restimulation assays were performed. <b><i>Results:</i></b> Whey- or casein-sensitized mice treated with αGalCer showed enhanced acute allergic skin reactions. The percentage of iNKT cells in the liver of sensitized mice was reduced compared to sham-sensitized mice. αGalCer treatment was found to deplete iNKT cells in the liver of sensitized as well as sham-sensitized mice, and these hepatocytes did not respond to ex vivo restimulation with αGalCer. αGalCer treatment did not reduce iNKT cell percentages in the spleen and MLN of sham-sensitized mice but abrogated the increase in iNKT cell percentage in the spleen upon whey sensitization, whereas it enhanced the iNKT cell percentage in the MLN of casein-sensitized mice. Due to the repeated application of αGalCer, livers were functionally depleted of iNKT cells. This resulted in an increased allergic effector response which was most pronounced in whey-sensitized mice and associated with enhanced whey-specific immunoglobulin levels. <b><i>Conclusion:</i></b> iNKT cells may suppress cow’s milk allergic symptoms in mice and may differentially regulate oral sensitization for casein and whey.