Human milk (HM) contains various bioactive antioxidants. Lactoferrin (Lf) has been assumed to be one of the major antioxidants in HM. We examined the antioxidative properties of iron-unsaturated human Lf (apo-hLf, the major form of Lf in HM) in two intestinal epithelial cell lines: (1) An intestinal epithelial cell line (IEC-6) were preincubated for 24 h with either 50 microg/mL of apo-hLf, iron-saturated human Lf (holo-hLf), iron-unsaturated bovine transferrin (apo-bTf), or 800 ng/mL of the iron-chelating compound deferoxamine (DFX), followed by hydrogen peroxide (H2O2) challenge to induce oxidative stress. Survival rates were significantly higher in the cells preincubated with apo-hLf and DFX than those preincubated with holo-hLf. (2) Caco-2 cells were preincubated with or without apo-hLf for 24 h, followed by an H2O2 challenge. Intracellular oxidative stress was assessed by a fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA). Fluorescent intensity of cell images and cell homogenates was significantly lower in the cells preincubated with apo-hLF than those preincubated without apo-hLF. Our study indicates that apo-hLf alleviates H2O2-induced oxidative damage in intestinal cells due to the iron-chelating capacity. Therefore, Lf in HM may act as an antioxidant in the gastrointestinal tract (GIT).