TccC3 and TccC5 from Photorhabdus luminescens are ADP-ribosyltransferases, which modify actin and Rho GTPases, respectively, thereby inducing polymerization and clustering of actin. The bacterial proteins are components of the Photorhabdus toxin complexes, consisting of the binding and translocation component TcdA1, a proposed linker component TcdB2 and the enzymatic component TccC3/5. While the action of the toxins on target proteins is clearly defined, uptake and translocation of the toxins into the cytosol of target cells are not well understood. Here we show by using pharmacological inhibitors that heat shock protein 90 (Hsp90) and peptidyl prolyl cis/trans isomerases (PPIases) including cyclophilins and FK506-binding proteins (FKBPs) facilitate the uptake of the ADP-ribosylating toxins into the host cell cytosol. Inhibition of Hsp90 and/or PPIases resulted in decreased intoxication of target cells by Photorhabdus toxin complexes determined by cell rounding and reduction of transepithelial electrical resistance of cell monolayers. ADP-ribosyltransferase activity of toxins and toxin-induced pore formation were notimpaired by the inhibitors of Hsp90 and PPIases. The Photorhabdus toxins interacted with Hsp90, FKBP51, Cyp40 and CypA, suggesting a role of these host cell factors in translocation and/or refolding of the ADP-ribosyltransferases.