The pig small intestinal brush border is a glycoprotein- and glycolipid-rich membrane that functions as a digestive/absorptive surface for dietary nutrients as well as a permeability barrier for pathogens. The present work was performed to identify carbohydrate-binding (lectin-like) proteins associated with the brush border. Chromatography on lactoseagarose was used to isolate such proteins, and their localization was studied biochemically and by immunofluorescence microscopy and immunogold electron microscopy. Immunoglobulin G (IgG) and immunoglobulin M (IgM) were the two major proteins isolated, indicating that naturally occurring anti-glycosyl antibodies are amongst the major lectin-like proteins in the gut. IgG, IgM as well as IgA were localized to the enterocyte brush border, and a brief lactose wash partially released all three immunoglobulins from the membrane, indicating that the anti-glycosyl antibodies constitute a major part of the immunoglobulins at the lumenal surface of the gut. The antibodies were associated with lipid rafts at the brush border and they frequently (52%) co-clustered with the raft marker galectin-4. A lactose wash increased the susceptibility of the brush border towards lectin PNA and cholera toxin B, suggesting that anti-glycosyl antibodies compete with other carbohydrate-binding proteins at the lumenal surface of the gut. Thus, anti-glycosyl antibodies constitute a major group of proteins associated with the enterocyte brush border membrane. We propose they function by protecting the lipid raft microdomains of the brush border against pathogens.