Milk fat globule epidermal growth factor-8 (MFG-E8) promotes phagocytic clearance of apoptotic cells to maintain normal tissue homeostasis. However, its functions in intestinal inflammatory disorders are unknown. Since the pathogenesis of those disorders are due to abnormal interactions between intestinal epithelial cells (IECs) and microbial pathogens, we analyzed the effects of MFG-E8 on IECs to determine its protective role in murine experimental colitis. Expression of αvβ3-integrin in Colon-26 cells was examined by RT-PCR and immunostaining. Colon-26 cells were pretreated with recombinant wild-type and mutant MFG-E8 proteins, following stimulation with flagellin as an inducer of innate immunity, and the effects of the recombinant proteins on inhibition of nuclear factor-κB (NF-κB) and inflammatory cytokine production in flagellin-stimulated Colon-26 cells were determined using a luciferase assay and EIA, respectively. Experimental colitis was induced in mice by intrarectal administration of trinitrobenzene sulfonic acid (TNBS). Recombinant proteins were then intrarectally administered into TNBS-induced colitic mice, after which disease activity parameters (body weight, colon length, histological score), as well as interleukin (IL)-6 and MIP-2 levels were determined in inflamed tissues. Flagellin-induced inflammatory cytokine production in vitro was significantly downregulated via αvβ3-integrin following pretreatment with wild-type MFG-E8 due to inhibition of NF-κB activation. In vivo, intrarectal treatment with wild-type MFG-E8, but not its mutant counterpart, significantly inhibited body weight loss, colon shortening and histological inflammation induced by TNBS administration. Our findings suggest that MFG-E8 has anti-inflammatory effects on flagellin-induced inflamed intestinal epithelial cells and may be a useful therapeutic agent for colitis.