Intestinal trefoil factor (TFF3/ITF), a member of trefoil factor family (TFF) domain peptides, is normally expressed by goblet cells and secreted into the lumen of the intestinal tract, and plays an important role in the maintenance and repair of the intestinal mucosal barrier. Significant amounts of TFF3/ITF are present in the meconium of human infants. Here, we describe a relatively simple method for extraction and purification of natural human TFF3/ITF from meconium. The purification methods include acid treatment, ammonium sulfate precipitation, isoelectric precipitation, and iron exchange chromatography. A relatively large amount of natural TFF3/ITF can be purified from meconium of full-term infants and this purified, natural TFF3/ITF is biologically active in an ethanol-induced rat gastric mucosal injury model.