Deoxynivalenol (DON) has critical health effects if the contaminated grains consumed by humans or animals. DON can have negative effects on the active transport of glucose and amino acids in the small intestine of chickens. As the underlying mechanisms are not fully elucidated, the present study was performed to delineate more precisely the effects of cycloheximide (protein synthesis inhibitor, CHX) and DON on the intestinal absorption of nutrients. This was to confirm whether DON effects on nutrient absorption are due to an inhibition of protein synthesis. Changes in ion transport and barrier function were assessed by short-circuit current (Isc) and transepithelial ion conductance (Gt) in Ussing chambers. Addition of D-glucose or L-glutamine to the luminal side of the isolated mucosa of the jejunum increased (P < 0.001) the Isc compared with basal conditions in the control tissues. However, the Isc was not increased by the glucose or glutamine addition after pre-incubation of tissues with DON or CHX. Furthermore, both DON and CHX reduced Gt, indicating that the intestinal barrier is compromised and consequently induced a greater impairment of the barrier function. The remarkable similarity between the activity of CHX and DON on nutrient uptake is consistent with their common ability to inhibit protein synthesis. It can be concluded that the decreases in transport activity by CHX was evident in this study using the chicken as experimental model. Similarly, DON has negative effects on the active transport of some nutrients, and these can be explained by its influence on protein synthesis.