Celiac disease (CD) is a multisystemic autoimmune inflammation of the intestinal tract induced by wheat gluten and related cereals in HLA-DQ2/8 positive individuals. An essential role in the pathogenesis of CD is played by a fraction of the peptic–tryptic digest of gluten, Frazer's Fraction (FF). Here, we investigate the effects of FF on the integrity of intestinal cells with particular emphasis on brush border membrane (BBM) components, their subsequent trafficking and endocytosis. Caco-2 cells were incubated with FF at different concentrations. Thereafter, several protein and lipid components of treated and untreated cells were analysed at the molecular, functional and cellular levels. The control employed tryptic–peptic digests of ovalbumin. Our results show that FF directly interacts with actin in an alternating manner eliciting substantial alterations in its integrity and extent in the BBM. These alterations lead to an impaired trafficking of SI to the apical membrane and reduction in its enzymatic function. ApN and DPPIV follow a transcytotic pathway and are only partly affected by FF. By contrast, the trafficking of LPH remains unaffected concomitant with its actin-independent trafficking pattern. Finally, the endocytic pathway is substantially blocked in FF-treated cells leading to an accumulation of cholesterol, and sphingolipids in the BBM. We conclude that FF deteriorates the actin cytoskeleton in Caco-2 leading to reduced protein sorting and hampered endocytic events with subsequent alterations in the protein and lipid composition of the BBM. The reduced levels of the disaccharidase SI in the BBM suggest a potential pathomechanism of carbohydrate malabsorption in CD.