Iota-toxin from Clostridium perfringens type E is a binary toxin consisting of two independent proteins, an enzymatic Ia and binding Ib component. Ia catalyses ADP-ribosylation of actin monomers, thus disrupting the actin cytoskeleton. In this report, we show that Ia plus Ib applied apically or basolaterally induce a rapid decrease in the transepithelial resistance (TER) of CaCo-2 cell monolayers and disorganization of actin filaments as well as the tight and adherens junctions. Ib alone, on the apical or basolateral side, slowly decreased the TER without affecting the actin cytoskeleton, possibly via pore formation. Interestingly, the two iota-toxin components inoculated separately on each cell surface induced cytopathic effects and a TER decrease. Anti-Ib sera, raised against the whole molecule or the Ia docking domain and applied to the opposite cell side versus Ib, neutralized the TER decrease. In addition, radioactive Ib incubated in the basolateral compartment was detected on the apical side by selective cell surface biotinylation. This argues for a transcytotic routing of Ib to mediate internalization of Ia from the opposite cell surface. Bafilomycin A1 also prevented the cytopathic effects of Ia and Ib applied separately to each cell side, possibly by blocking translocation of Ia into the cytosol and/or the intracellular transport of Ib. Ib is either routed into the cell independently of Ia, trans-cytosed and permanently exposed on the opposite cell surface or continuously recycled between an endosomal compartment and the cell surface.