Clorpyrifos (CPF), one of the most widely used organophosphorothionate pesticide can be detected as residues in food and drinking water; therefore the oral route is the major route of exposure for the general population, including children, following household use of this insecticide. Aim of this work was to investigate the possible acute cytotoxic effects of CPF on intestine and the integrity of the epithelial barrier, using Caco-2/TC7 cells as intestinal in vitro model. High level of CPF found inside the cells, corresponding to about 80% of the nominal concentration tested (30, 50 and 250microM), chosen as representative of the concentrations attainable in the intestinal lumen after actual levels of human oral exposure. In these conditions, no cytotoxicity in terms of cellular viability was observed. However, at the highest CPF nominal concentration (250microM) the impairment of barrier integrity was evidenced, due only to the parent compound, since no CPF metabolites could be detected in our experimental conditions. CPF itself was demonstrated to interfere with the tight junction on this in vitro model of epithelial intestinal cells, altering the barrier integrity and very likely the absorption of other co-administered chemicals.