Deoxynivalenol (DON) is a mycotoxin of the trichothecenes family to which human exposure levels can be high. Epidemiological studies suggest a link between DON and gastrointestinal illness. We investigated the interaction of DON with Caco-2 cells, a widely used in vitro model of the human intestinal barrier. The apical to basolateral (absorption) and basolateral to apical (excretion) transports of DON were found strictly proportional to both the initial concentration and the duration of the incubation. The absorption and excretion mean rates were similar to those of mannitol and were increased in the presence of EGTA, a calcium chelator. These data suggest that DON crosses the intestinal mucosa by a paracellular pathway through the tight junctions although some passive transcellular diffusion may not be ruled out. The DON transport was not affected by P-glycoprotein (PgP) or multidrug resistance-associated proteins (MRPs) inhibitors. A prolonged exposure to DON provokes the phosphorylation of the mitogen-activated protein kinases (MAPKs) Erk1/2, p38 and SAPK/JNK, as well as a decrease of the transepithelial resistance, suggesting that DON could trigger intestinal inflammation. These data imply that a chronic exposure to DON contaminated foods may negatively affect human health by altering the intestinal mucosa integrity and by inducing the MAPKs implicated in inflammation.