Dietary deoxynivalenol (DON) impairs the intestinal immune system and digestive functions of broiler chickens. However, little is known whether increasing doses of DON similarly affect the intestinal functions in different segments of the small intestine in chickens and whether a second oral challenge may potentiate those effects. The present objective was to investigate the effect of increasing dietary DON concentrations on the relative expression of genes for tight junction proteins, mucins, toll-like receptors (TLR), and cytokines in duodenum and jejunum, jejunal mucosal permeability, as well as on α-1-acid glycoprotein and IgA in serum with or without an additional oral lipopolysaccharide (LPS) challenge. Eighty 1-d-old chickens were fed diets with increasing DON concentrations (0, 2.5, 5, and 10 mg DON/kg diet) for 5 wk. One day before sampling, half of the chickens received an oral challenge with 1-mg Escherichia coli O55:B5 LPS/kg BW. Ussing chambers were used to measure the jejunal permeability in birds receiving 10-mg DON/kg feed with or without LPS challenge and 0-mg DON/kg feed without LPS. Increasing DON concentrations of up to 5-mg DON/kg increased (P < 0.05) the duodenal expression of TLR2, IL6, and Claudin 1 (CLDN1) by up to 84%, 88%, and 48%, respectively, compared with the noncontaminated diet. Likewise, jejunal CLDN1 expression increased up to 23% in the chickens fed DON concentrations of up to 5-mg DON/kg diet (P < 0.05). Moreover, increasing DON concentrations linearly and quadratically decreased (P < 0.05) the jejunal expression of TLR2 and transforming growth factor-β 1, respectively. The additional LPS challenge increased (P < 0.040) duodenal occludin expression by 10% as well as the jejunal tissue conductance in chickens of the 10 DON group (P = 0.050). In conclusion, dietary DON differently affected the duodenal and jejunal expression of genes for tight-junction proteins and proinflammatory signaling pathways. The additional LPS challenge did not potentiate the DON effect but it seemed to induce a certain up-regulation of the proinflammatory response in the duodenum and enhanced the mucosal permeability in the jejunum.