In recent years, the deleterious effects attributed to mycotoxins, in particular on the intestine, faced increased attention and it was shown that deoxynivalenol (DON) causes adverse effects on gut health. In this context, it has been repeatedly reported that DON can alter the intestinal morphology, disrupt the intestinal barrier and reduce nutrient absorption. The underlying mechanism of a compromised intestinal barrier caused by DON in chickens has yet to be illustrated. Although, DON is rapidly absorbed from the upper parts of the small intestine, the effects on the large intestine cannot be excluded. Additionally, a damaging effect of DON on the gut epithelium might decrease the resistance of the gut against infectious agents. Consequently, the objectives of the present studies were: (1) to investigate the impact of DON on the epithelial paracellular permeability by demonstrating the mucosal to serosal flux of 14C-mannitol in the small and large intestine applying Ussing chambers and (2) to delineate the effects of DON on the colonization and translocation of Escherichia coli. Both parameters are well suited as potential indicators for gut barrier failure. For this, a total of 75 one-day-old Ross 308 broiler chickens were housed in floor pens on wood shavings with feed and water provided ad libitum. Birds were randomly allocated to three different groups (n = 25 with 5 replicates/group) and were fed for 5 weeks with either contaminated diets (5 or 10 mg DON/kg feed) or basal diets (control). Body weight (BW) and BW gain of birds in the group fed with 10 mg/kg DON were significantly lower than in group with 5 mg/kg DON and the control group. Moreover, the mannitol flux in jejunum and cecum was significantly (P < 0.05) higher in DON-fed groups compared to control birds. Consistent with this, DON enhanced the translocation of E. coli with a higher number of bacteria encountered in the spleen and liver. Altogether, the actual results verified that DON can alter the intestinal paracellular permeability in broiler chickens and facilitates the translocation of enteric microorganisms such as E. coli to extra-intestinal organs. Considering that moderate levels of DON are present in feed, the consumption of DON-contaminated feed can induce an intestinal breakdown with negative consequences on broiler health.