ADP-ribosylation of Rho(A,B,C) by the family of exoenzyme C3-like transferases induces reorganization of the actin cytoskeleton based on inactivation of RhoA. No data are available on the role of RhoB in C3-treated cells. In murine fibroblasts treated with the cell-permeable exoenzyme C3 from Clostridium limosum (C3), an increase in the level of RhoB was observed. This upregulation of RhoB was based on transcriptional activation, as it was responsive to inhibition by actinomycin D and accompanied by activation of the rhoB promoter. Upregulation of RhoB was not observed in cells treated with either the actin ADP-ribosylating C2 toxin from Clostridium botulinum or latrunculin B, suggesting that inactivation of Rho but not actin reorganization was required for the upregulation of RhoB. This notion was confirmed, as the Rho/Rac/Cdc42-glucosylating toxin B from Clostridium difficile (TcdB) but not the Rac/R-Ras-glucosylating variant toxin B from C. difficile strain 1470 serotype F (TcdBF) induced a strong upregulation of RhoB. Upregulation of RhoB was further observed in response to the Rac/(H-,K-,N-,R-)Ras-glucosylating lethal toxin from Clostridium sordellii. The level of active, GTP-bound RhoB was increased in TcdB-treated cells compared to untreated cells (as determined by Rhotekin pull-down assay). In contrast, no active RhoB was found in C3-treated cells. RhoB-GTP was required for the TcdB-induced apoptosis (cytotoxic effect), as this effect was responsive to inhibition by C3. In conclusion, RhoB was upregulated by Rho-/Ras-inactivating toxins, as a consequence of the inactivation of either Rho(A,B,C) or (H-,K-,N-)Ras. In TcdB-treated cells, RhoB escaped its inactivation and was required for the cytotoxic effect.