Clostridium perfringens TpeL belongs to a family of large clostridial glucosylating cytotoxins. TpeL modifies Rac1 and Ras subfamily proteins. Herein we report TpeL-induced formation of stress fibers via RhoA-Rho kinase (ROCK) signaling. A recombinant protein (TpeL1–525) derived from the TpeL N-terminal catalytic domain in the presence of streptolysin O (SLO) induced the formation of actin stress fibers in Madin–Darby canine kidney (MDCK) cells in a dose-dependent manner. The RhoA/ROCK pathway is known to control the formation of stress fibers. We examined the role of the RhoA/ROCK pathway in TpeL-induced formation of stress fibers. TpeL1–525-induced formation of stress fibers was inhibited by the ROCK inhibitor, Y27632 and Rho protein inhibitor, C3 transferase. TpeL1–525 activated RhoA and ROCK in a dose-dependent manner. C3 transferase blocked TpeL1–525-induced activation of RhoA and ROCK whereas Y27632 inhibited TpeL-induced activation of ROCK. These results demonstrate for the first time that TpeL induces the formation of stress fibers by activating the RhoA/ROCK signaling pathway.