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 (lectinlike) proteins associated with the brush border. Chromatography on lactose-agarose was used to isolate such proteins, and their localization was studied biochemically and by immunofluorescence microscopy and immunogold electron microscopy. IgG and IgM were the two major proteins isolated, indicating that naturally occurring anti-glycosyl antibodies are among the major lectinlike proteins in the gut. IgG and 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 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%) coclustered with the raft marker galectin 4. A lactose wash increased the susceptibility of the brush border toward lectin peanut agglutin 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.