Characterization and identification of peptides with bioactivity from food have received considerable interest recently since such bioactive components must be adequately documented if they are part of functional food claims. We have characterized peptides from colostrum or those generated by a simulated gastrointestinal digest (GI) and tested them for bioactivity using murine intestinal (mICc12) cells and compared with bioactivity of intact colostrum. The peptides were recovered in the permeate after dialysis. The presence of peptides in the permeate was confirmed by C18 RP-HPLC, determination of free amino termini and MALDI MS. The bioactivity of the intact colostrum and colostral peptides in the permeate was tested using mICc12 cells stimulated in the absence or presence of different bacterial ligands that mediate cellular activation through stimulation of Toll-like receptors (TLR). Whereas intact colostrum generally reduced TLR-mediated signaling, the isolated peptides seemed to either stimulate or reduce the immune response depending on the bacterial ligand used for stimulation. Interestingly, the most potent bioactive peptides originated from nondigested colostrum, which had only been subject to endogenous protease activity. Identified peptides in the nondigested colostrum originated exclusively from the casein fraction of colostrum as shown by MALDI MS/MS identification. Thus, multiple components with different bioactivities towards the innate immune response appear in bovine colostrum. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.